Engineered transferrin receptor binding polypeptide

Abstract

To provide polypeptides that bind to a transferrin receptor.SOLUTION: Provided herein are polypeptides that bind to a transferrin receptor, methods of producing such polypeptides, and methods of using the polypeptides to target a composition to a transferrin receptor-expressing cell.SELECTED DRAWING: None

Description

[Background technology]

[0001] Background of the Invention Various methods have been developed to engineer proteins to bind to targets to which they normally do not bind. For example, libraries can be generated and screened for proteins engineered with desired binding or enzymatic activity.

[0002] The transferrin receptor is a transferrin transport protein that is required for intracellular iron transport, among other functions, and is regulated according to intracellular iron concentration. Transferrin receptors are expressed in endothelia, including those of the blood-brain barrier, and are expressed at high levels in various cancer cells and inflammatory cells. Transferrin is one of the receptors that mediate transcytosis of its cognate ligand across the blood-brain barrier. Therefore, the transferrin receptor may be a desirable target for introducing substances into cells by intracellular endocytosis or transcellular transcytosis. Summary of the Invention

[0003] The present inventors have developed polypeptides containing CH2 and CH3 domains capable of binding to the transferrin receptor (TfR). These polypeptides have been engineered with substitutions within the CH2 or CH3 domains that create novel TfR-binding sites. TfR is highly expressed at the blood-brain barrier (BBB), where TfR naturally transports transferrin from the blood into the brain. Because these polypeptides bind to TfR, they can also be transported across the BBB and can further be used to transport conjugated therapeutic agents (e.g., therapeutic polypeptides, antibody variable regions such as Fabs, and small molecules) across the BBB. This approach can significantly improve the brain uptake of therapeutic agents and is therefore extremely useful for treating disorders and diseases where delivery to the brain is beneficial.

[0004] In one aspect, a polypeptide is provided that comprises a modified CH3 domain that specifically binds to the transferrin receptor, wherein the modified CH3 domain comprises four, five, six, seven, eight, or nine substitutions at a set of amino acid positions including 157, 159, 160, 161, 162, 163, 186, 189, and 194, wherein the substitutions and positions are determined with reference to amino acids 114-220 of SEQ ID NO:1. In some embodiments, the modified CH3 domain further comprises one, two, three, or four substitutions at positions including 153, 164, 165, and 188. In some embodiments, the polypeptide binds to the apical domain of the transferrin receptor. In some embodiments, the polypeptide binds to the transferrin receptor without inhibiting transferrin binding to the transferrin receptor. In some embodiments, the polypeptide binds to an epitope comprising amino acid 208 of the transferrin receptor sequence.

[0005] In some embodiments, the modified CH3 domain comprises a Trp at position 161. In some embodiments, the modified CH3 domain comprises an aromatic amino acid at position 194. In some embodiments, the aromatic amino acid at position 194 is Trp or Phe.

[0006] In some embodiments, the modified CH3 domain comprises at least one position selected from the following: Leu, Tyr, Met, or Val at position 157, Leu, Thr, His, or Pro at position 159, Val, Pro, or an acidic amino acid at position 160, Trp or Gly at position 161, Val, Ser, or Ala at position 162, Glu, Ala, Ser, Leu, Thr, or Pro at position 186, Thr, or an acidic amino acid at position 189, and Trp, Tyr, His, or Phe at position 194. In some embodiments, the modified CH3 domain comprises two, three, four, five, six, seven, or eight positions selected from the following: that is, Leu, Tyr, Met, or Val at position 157, Leu, Thr, His, or Pro at position 159, Val, Pro, or an acidic amino acid at position 160, Trp or Gly at position 161, Val, Ser, or Ala at position 162, Glu, Ala, Ser, Leu, Thr, or Pro at position 186, Thr, or an acidic amino acid at position 189, and Trp, Tyr, His, or Phe at position 194. In some embodiments, the engineered CH3 domain comprises Leu or Met at position 157, Leu, His, or Pro at position 159, Val at position 160, Trp or Gly at position 161, Val or Ala at position 162, Pro at position 186, Thr at position 189, and / or Trp at position 194.

[0007] In some embodiments, the modified CH3 domain comprises at least one position selected from the following: Leu, Tyr, Met, or Val at position 157, Leu, Thr, His, or Pro at position 159, Val, Pro, or an acidic amino acid at position 160, Trp at position 161, Val, Ser, or Ala at position 162, Glu, Ala, Ser, Leu, Thr, or Pro (e.g., Thr or Pro) at position 186, Thr or an acidic amino acid at position 189, and Trp, Tyr, His, or Phe at position 194. In some embodiments, the modified CH3 domain comprises two, three, four, five, six, seven, or eight positions selected from the following: that is, Leu, Tyr, Met, or Val at position 157, Leu, Thr, His, or Pro at position 159, Val, Pro, or an acidic amino acid at position 160, Trp at position 161, Val, Ser, or Ala at position 162, Glu, Ala, Ser, Leu, Thr, or Pro (e.g., Thr or Pro) at position 186, Thr or an acidic amino acid at position 189, and Trp, Tyr, His, or Phe at position 194. In some embodiments, the engineered CH3 domain comprises Leu or Met at position 157, Leu, His, or Pro at position 159, Val at position 160, Trp at position 161, Val or Ala at position 162, Pro at position 186, Thr at position 189, and / or Trp at position 194.

[0008] In some embodiments, the modified CH3 domain further comprises Ser, Thr, Gln, or Phe at position 164. In some embodiments, the modified CH3 domain further comprises Trp, Tyr, Leu, or Gln at position 153. In some embodiments, the modified CH3 domain further comprises Gln, Phe, or His at position 165. In some embodiments, the modified CH3 domain further comprises Trp at position 153 and / or Gln at position 165. In some embodiments, the modified CH3 domain further comprises Glu at position 188.

[0009] In some embodiments, the modified CH3 domain comprises a Tyr at position 157, a Thr at position 159, a Glu or Val at position 160, a Trp at position 161, a Ser at position 162, a Ser or Thr at position 186, a Glu at position 189, and / or a Phe at position 194. In some embodiments, the modified CH3 domain further comprises a Trp, Tyr, Leu, or Gln at position 153. In some embodiments, the modified CH3 domain further comprises a Glu at position 188. In some embodiments, the modified CH3 domain further comprises a Trp at position 153 and / or a Glu at position 188. In some embodiments, the modified CH3 domain further comprises a Leu at position 153 and / or a Glu at position 188. In some embodiments, the modified CH3 domain comprises an Asn at position 163.

[0010] In some embodiments, the modified CH3 domain comprises one or more of the following substitutions: Trp at position 153, Thr at position 159, Trp at position 161, Val at position 162, Ser or Thr at position 186, Glu at position 188, and / or Phe at position 194.

[0011] In some embodiments, the modified CH3 domain further comprises one, two, or three positions selected from the following: Lys, Arg, Gly, or Pro at position 187; Ser, Thr, Glu, or Lys at position 197; and Ser, Trp, or Gly at position 199.

[0012] In some embodiments, the modified CH3 domain has at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 114-220 of any one of SEQ ID NOs: 4-29, 236-299, and 422-435. In some embodiments, the modified CH3 domain has at least 85% identity to amino acids 114-220 of SEQ ID NO: 1, with the proviso that the percent identity does not include the set of positions 157, 159, 160, 161, 162, 163, 186, 189, and 194. In some embodiments, the modified CH3 domain comprises amino acids 157-163 and / or 186-194 of any one of SEQ ID NOs: 4-29, 236-299, and 422-435.

[0013] In some embodiments, the modified CH3 domain comprises at least one position selected from the following: Trp, Leu, or Glu at position 153; Tyr or Phe at position 157; Thr at position 159; Glu at position 160; Trp at position 161; Ser, Ala, Val, or Asn at position 162; Ser or Asn at position 163; Thr or Ser at position 186; Glu or Ser at position 188; Glu at position 189; and Phe at position 194. In some embodiments, the modified CH3 domain comprises two, three, four, five, six, seven, eight, nine, ten, or eleven positions (e.g., eleven positions) selected from: Trp, Leu, or Glu (e.g., Trp or Leu) at position 153, Tyr or Phe at position 157, Thr at position 159, Glu at position 160, Trp at position 161, Ser, Ala, Val, or Asn at position 162, Ser or Asn at position 163, Thr or Ser at position 186, Glu or Ser at position 188, Glu at position 189, and Phe at position 194. In some embodiments, the modified CH3 domain has at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs:236-299 and 422-435.

[0014] In some embodiments, the modified CH3 domain comprises the following amino acids: Trp, Leu, or Glu (e.g., Trp or Leu) at position 153, Tyr or Phe at position 157, Thr at position 159, Glu at position 160, Trp at position 161, Ser, Ala, Val, or Asn at position 162, Ser or Asn at position 163, Thr or Ser at position 186, Glu or Ser at position 188, Glu at position 189, and Phe at position 194. In further embodiments, the modified CH3 domain has at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs:236-299 and 422-435.

[0015] In some embodiments, the modified CH3 domain comprises amino acids 153-163 and / or 186-194 of any one of SEQ ID NOs: 236-299 and 422-435.

[0016] In some embodiments, provided herein are polypeptides comprising a modified CH3 domain that specifically binds to a transferrin receptor, wherein the modified CH3 domain has at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 114-220 of any one of SEQ ID NOs: 4-29, 236-299, and 422-435. In certain embodiments, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 (e.g., 11-16) residues at positions corresponding to 153, 157, 159, 160, 161, 162, 163, 164, 165, 186, 187, 188, 189, 194, 197, and 199 relative to SEQ ID NO:1 are not deleted or substituted in SEQ ID NOs:4-29 or 236-299.

[0017] In any of the above embodiments, the modified CH3 domain further comprises (i) a Trp at position 139 (T139W), or (ii) a Ser at position 139 (T139S), an Ala at position 141 (L141A), and a Val at position 180 (Y180V), where the amino acid positions are determined with reference to SEQ ID NO: 1. In any of the above embodiments, the modified CH3 domain further comprises (i) a Leu at position 201 (M201L) and a Ser at position 207 (N207S), or (ii) a Ser or Ala at position 207 (N207S or N207A), where the amino acid positions are determined with reference to SEQ ID NO: 1.

[0018] In another aspect, the disclosure features a polypeptide including a modified CH3 domain that specifically binds to a transferrin receptor, wherein the modified CH3 domain includes one or more substitutions at a set of amino acid positions including: 153, 157, 159, 160, 162, 163, 186, 188, 189, 194, 197, and 199, wherein the substitutions and the positions are determined with reference to the sequence of SEQ ID NO:13. In some embodiments, the modified CH3 domain comprises Glu, Leu, Ser, Val, Trp, or Tyr at position 153, an aromatic amino acid, Met, Pro, or Val at position 157, Thr, Asn, or Val at position 159, Glu, He, Pro, or Val at position 160, an aliphatic amino acid, Ser, or Thr at position 162, Ser, Asn, Arg, or Thr at position 163, Thr, His, or Ser at position 186, Glu, Ser, Asp, Gly, Thr, Pro, Gln, or Arg at position 188, Glu or Arg at position 189, Phe, His, Lys, Tyr, or Trp at position 194, Ser, Thr, or Trp at position 197, and Ser, Cys, Pro, Met, or Trp at position 199. In specific embodiments, the aromatic amino acid at position 157 is Tyr, Phe, or Trp, and the aliphatic amino acid at position 162 is Ala, Ile, or Val.

[0019] In some embodiments, the engineered CH3 domain comprises Glu, Leu, or Trp at position 153, an aromatic amino acid at position 157, Thr at position 159, Glu at position 160, an aliphatic amino acid or Ser at position 162, Ser or Asn at position 163, Thr or Ser at position 186, Glu or Ser at position 188, Glu at position 189, Phe, His, Tyr, or Trp at position 194, Ser at position 197, and Ser at position 199. In a specific embodiment, the aromatic amino acid at position 157 is Tyr or Phe, and the aliphatic amino acid at position 162 is Ala or Val.

[0020] In some embodiments, the modified CH3 domain has the sequence of SEQ ID NO:556 or 559.

[0021] In some embodiments, the modified CH3 domain comprises a substitution at a set of amino acid positions including: 153, 157, 159, 160, 162, 163, 186, 188, 189, 194, 197, and 199. In specific embodiments, the modified CH3 domain has the sequence of any one of SEQ ID NOs:563-574.

[0022] In some embodiments, the modified CH3 domain comprises Glu, Leu, or Trp at position 153, Tyr or Phe at position 157, Thr at position 159, Glu at position 160, Ala, Val, or Ser at position 162, Ser or Asn at position 163, Thr or Ser at position 186, Glu or Ser at position 188, Glu at position 189, Phe at position 194, Ser at position 197, and Ser at position 199. In a specific embodiment, the modified CH3 domain has the sequence of SEQ ID NO:562.

[0023] In another aspect, the disclosure features a polypeptide including a modified CH3 domain that specifically binds to a transferrin receptor, wherein the modified CH3 domain includes one or more substitutions at a set of amino acid positions including: 153, 157, 159, 160, 162, 163, 164, 186, 189, and 194, wherein the substitutions and the positions are determined with reference to the sequence of SEQ ID NO:9. In some embodiments, the modified CH3 domain comprises Glu or Trp at position 153, Val, Trp, Leu, or Tyr at position 157, Leu, Pro, Phe, Thr, or His at position 159, Pro, Val, or Glu at position 160, Ala, Ser, Val, or Gly at position 162, Leu, His, Gln, Gly, Val, Ala, Asn, Asp, Thr, or Glu at position 163, Thr, Phe, Gln, Val, or Tyr at position 164, Leu, Ser, Glu, Ala, or Pro at position 186, Glu, Asp, Thr, or Asn at position 189, and Trp, Tyr, Phe, or His at position 194.

[0024] In some embodiments, the modified CH3 domain comprises Glu or Trp at position 153, Trp, Leu, or Tyr at position 157, Thr or His at position 159, Val at position 160, Ala, Ser, or Val at position 162, Val, Asn, or Thr at position 163, Gln or Tyr at position 164, Pro at position 186, Thr or Asn at position 189, and Trp, Tyr, Phe, or His at position 194. In specific embodiments, the modified CH3 domain has the sequence of SEQ ID NO:577 or 580.

[0025] In another aspect, provided are polypeptides comprising a modified CH3 domain that specifically binds to a transferrin receptor, wherein the modified CH3 domain comprises four, five, six, seven, or eight substitutions at the set of amino acid positions comprising: 118, 119, 120, 122, 210, 211, 212, and 213, wherein the substitutions and positions are determined with reference to amino acids 114-220 of SEQ ID NO: 1. In some embodiments, the modified CH3 domain comprises a Gly at position 210, a Phe at position 211, and / or an Asp at position 213. In some embodiments, the modified CH3 domain comprises at least one position selected from the following: Phe or Ile at position 118; Asp, Glu, Gly, Ala, or Lys at position 119; Tyr, Met, Leu, Ile, or Asp at position 120; Thr or Ala at position 122; Gly at position 210; Phe at position 211; His, Tyr, Ser, or Phe at position 212; and Asp at position 213. In some embodiments, the modified CH3 domain comprises two, three, four, five, six, seven, or eight positions selected from the following: Phe or Ile at position 118, Asp, Glu, Gly, Ala, or Lys at position 119, Tyr, Met, Leu, Ile, or Asp at position 120, Thr or Ala at position 122, Gly at position 210, Phe at position 211, His, Tyr, Ser, or Phe at position 212, and Asp at position 213. In some embodiments, the modified CH3 domain has at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 114-220 of any one of SEQ ID NOs:30-46. In some embodiments, the modified CH3 domain has at least 85% identity to amino acids 114-220 of SEQ ID NO:1, with the proviso that the percent identity does not include the set of positions 118, 119, 120, 122, 210, 211, 212, and 213. In some embodiments, the modified CH3 domain comprises amino acids 118-122 and / or 210-213 of any one of SEQ ID NOs:30-46.In some embodiments, the modified CH3 domain further comprises (i) a Trp at position 139 (T139W), or (ii) a Ser at position 139 (T139S), an Ala at position 141 (L141A), and a Val at position 180 (Y180V), where the amino acid positions are determined with reference to SEQ ID NO:1. In some embodiments, the modified CH3 domain further comprises (i) a Leu at position 201 (M201L) and a Ser at position 207 (N207S), or (ii) a Ser or Ala at position 207 (N207S or N207A), where the amino acid positions are determined with reference to SEQ ID NO:1.

[0026] In some embodiments, the corresponding unmodified CH3 domain is a CH3 domain of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the polypeptide is linked to a CH2 domain (e.g., a CH2 domain of IgG1, IgG2, IgG3, or IgG4). In some embodiments, the CH2 domain contains one or both of the following sets of modifications relative to the amino acid sequence of SEQ ID NO:1: (a) Ala at positions 7 and 8 (L7A and L8A), and (b) Tyr at position 25 (M25Y), Thr at position 27 (S27T), and Glu at position 29 (T29E). In some embodiments, set (a) further comprises a Gly at position 102 (P102G). In some embodiments, the polypeptide is further linked to a Fab via a hinge region. In some embodiments, the Fab binds to a tau protein (e.g., a human tau protein) or a fragment thereof. The tau protein may be a phosphorylated tau protein, a non-phosphorylated tau protein, a splice isoform of tau protein, an N-terminally truncated tau protein, a C-terminally truncated tau protein, and / or a fragment thereof. In some embodiments, the Fab binds to a beta-secretase 1 (BACE1) protein (e.g., a human BACE1 protein) or a fragment thereof. The BACE1 protein may be a splice isoform of the BACE1 protein or a fragment thereof. In some embodiments, the Fab binds to a triggering receptor expressed on myeloid cells 2 (TREM2) protein (e.g., a human TREM2 protein) or a fragment thereof. In some embodiments, the Fab binds to an alpha-synuclein protein (e.g., a human alpha-synuclein protein) or a fragment thereof. The alpha-synuclein protein may be monomeric alpha-synuclein, oligomeric alpha-synuclein, alpha-synuclein fibrils, soluble alpha-synuclein, and / or a fragment thereof. In some embodiments, the polypeptide is the first polypeptide of a dimer such that the dimer is monovalent for binding to the transferrin receptor.In some embodiments, the polypeptide is a first polypeptide that binds to the transferrin receptor and dimerizes with a second polypeptide that comprises a modified CH3 domain, hi some embodiments, the modified CH3 domain of the second polypeptide is the same as the modified CH3 domain of the first polypeptide.

[0027] In another aspect, provided are polypeptides comprising a modified CH2 domain that specifically binds to a transferrin receptor, wherein the modified CH2 domain comprises 4, 5, 6, 7, 8, or 9 substitutions at the set of amino acid positions comprising: 47, 49, 56, 58, 59, 60, 61, 62, and 63, wherein the substitutions and positions are determined with reference to amino acids 4-113 of SEQ ID NO: 1. In some embodiments, the modified CH2 domain comprises Glu at position 60 and / or Trp at position 61. In some embodiments, the modified CH2 domain comprises at least one position selected from the following: Glu, Gly, Gln, Ser, Ala, Asn, Tyr, or Trp at position 47; Ile, Val, Asp, Glu, Thr, Ala, or Tyr at position 49; Asp, Pro, Met, Leu, Ala, Asn, or Phe at position 56; Arg, Ser, Ala, or Gly at position 58; Tyr, Trp, Arg, or Val at position 59; Glu at position 60; Trp or Tyr at position 61; Gln, Tyr, His, Ile, Phe, Val, or Asp at position 62; and Leu, Trp, Arg, Asn, Tyr, or Val at position 63. In some embodiments, the modified CH2 domain comprises at least two, three, four, five, six, seven, eight, or nine positions selected from the following: Glu, Gly, Gln, Ser, Ala, Asn, Tyr, or Trp at position 47; Ile, Val, Asp, Glu, Thr, Ala, or Tyr at position 49; Asp, Pro, Met, Leu, Ala, Asn, or Phe at position 56; Arg, Ser, Ala, or Gly at position 58; Tyr, Trp, Arg, or Val at position 59; Glu at position 60; Trp or Tyr at position 61; Gln, Tyr, His, Ile, Phe, Val, or Asp at position 62; and Leu, Trp, Arg, Asn, Tyr, or Val at position 63.In some embodiments, the modified CH2 domain comprises Glu, Gly, Gln, Ser, Ala, Asn, or Tyr at position 47, Ile, Val, Asp, Glu, Thr, Ala, or Tyr at position 49, Asp, Pro, Met, Leu, Ala, or Asn at position 56, Arg, Ser, or Ala at position 58, Tyr, Trp, Arg, or Val at position 59, Glu at position 60, Trp at position 61, Gln, Tyr, His, Ile, Phe, or Val at position 62, and / or Leu, Trp, Arg, Asn, or Tyr at position 63.

[0028] In some embodiments, the modified CH2 domain comprises Arg at position 58, Tyr or Trp at position 59, Glu at position 60, Trp at position 61, and / or Arg or Trp at position 63. In some embodiments, the modified CH2 domain has at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 4-113 of any one of SEQ ID NOs:47-62. In some embodiments, the modified CH2 domain has at least 85% identity to amino acids 4-113 of SEQ ID NO:1, provided that the set of positions 47, 49, 56, 58, 59, 60, 61, 62, and 63 are not included in the percent identity. In some embodiments, the modified CH2 domain comprises amino acids 47-49 and / or 56-63 of any one of SEQ ID NOs:47-62.

[0029] In another aspect, provided are polypeptides comprising a modified CH2 domain that specifically binds to a transferrin receptor, wherein the modified CH2 domain comprises 4, 5, 6, 7, 8, 9, or 10 substitutions at the set of amino acid positions comprising: 39, 40, 41, 42, 43, 44, 68, 70, 71, and 72, wherein the substitutions and positions are determined with reference to amino acids 4-113 of SEQ ID NO: 1. In some embodiments, the modified CH2 domain comprises Pro at position 43, Glu at position 68, and / or Tyr at position 70. In some embodiments, the modified CH2 domain comprises at least one position selected from the following: Pro, Phe, Ala, Met, or Asp at position 39; Gln, Pro, Arg, Lys, Ala, Ile, Leu, Glu, Asp, or Tyr at position 40; Thr, Ser, Gly, Met, Val, Phe, Trp, or Leu at position 41; Pro, Val, Ala, Thr, or Asp at position 42; Pro, Val, or Phe at position 43; Trp, Gln, Thr, or Glu at position 44; Glu, Val, Thr, Leu, or Trp at position 68; Tyr, His, Val, or Asp at position 70; Thr, His, Gln, Arg, Asn, or Val at position 71; and Tyr, Asn, Asp, Ser, or Pro at position 72. In some embodiments, the modified CH2 domain has one of the following residues: Pro, Phe, Ala, Met, or Asp at position 39; Gln, Pro, Arg, Lys, Ala, Ile, Leu, Glu, Asp, or Tyr at position 40; Thr, Ser, Gly, Met, Val, Phe, Trp, or Leu at position 41; Pro, Val, Ala, Thr, or Asp at position 42; and Pro, Val, or or Phe at position 44; Trp, Gln, Thr, or Glu at position 44; Glu, Val, Thr, Leu, or Trp at position 68; Tyr, His, Val, or Asp at position 70; Thr, His, Gln, Arg, Asn, or Val at position 71; and Tyr, Asn, Asp, Ser, or Pro at position 72.

[0030] In some embodiments, the modified CH2 domain comprises Pro, Phe, or Ala at position 39, Gln, Pro, Arg, Lys, Ala, or Ile at position 40, Thr, Ser, Gly, Met, Val, Phe, or Trp at position 41, Pro, Val, or Ala at position 42, Pro at position 43, Trp or Gln at position 44, Glu at position 68, Tyr at position 70, Thr, His, or Gln at position 71, and / or Tyr, Asn, Asp, or Ser at position 72. In some embodiments, the modified CH2 domain comprises Met at position 39, Leu or Glu at position 40, Trp at position 41, Pro at position 42, Val at position 43, Thr at position 44, Val or Thr at position 68, His at position 70, His, Arg, or Asn at position 71, and / or Pro at position 72. In some embodiments, the modified CH2 domain comprises an Asp at position 39, an Asp at position 40, a Leu at position 41, a Thr at position 42, a Phe at position 43, a Gln at position 44, a Val or Leu at position 68, a Val at position 70, a Thr at position 71, and / or a Pro at position 72.

[0031] In some embodiments, the modified CH2 domain has at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 4-113 of any one of SEQ ID NOs:63-85. In some embodiments, the modified CH2 domain has at least 85% identity to amino acids 4-113 of SEQ ID NO:1, with the proviso that the percent identity does not include the set of positions 39, 40, 41, 42, 43, 44, 68, 70, 71, and 72. In some embodiments, the modified CH2 domain comprises amino acids 39-44 and / or 68-72 of any one of SEQ ID NOs:63-85.

[0032] In another aspect, a polypeptide is provided that comprises a modified CH2 domain that specifically binds to a transferrin receptor, wherein the modified CH2 domain comprises 4, 5, 6, 7, 8, 9, or 10 substitutions at a set of amino acid positions including positions 41, 42, 43, 44, 45, 65, 66, 67, 69, and 73, wherein the substitutions and positions are determined relative to amino acids 4-113 of SEQ ID NO:1. In some embodiments, the modified CH2 domain comprises at least one position selected from the following: Val or Asp at position 41; Pro, Met, or Asp at position 42; Pro or Trp at position 43; Arg, Trp, Glu, or Thr at position 44; Met, Tyr, or Trp at position 45; Leu or Trp at position 65; Thr, Val, Ile, or Lys at position 66; Ser, Lys, Ala, or Leu at position 67; His, Leu, or Pro at position 69; and Val or Trp at position 73. In some embodiments, the modified CH2 domain comprises two, three, four, five, six, seven, eight, nine, or ten positions selected from the following: Val or Asp at position 41; Pro, Met, or Asp at position 42; Pro or Trp at position 43; Arg, Trp, Glu, or Thr at position 44; Met, Tyr, or Trp at position 45; Leu or Trp at position 65; Thr, Val, Ile, or Lys at position 66; Ser, Lys, Ala, or Leu at position 67; His, Leu, or Pro at position 69; and Val or Trp at position 73.

[0033] In some embodiments, the modified CH2 domain comprises Val at position 41, Pro at position 42, Pro at position 43, Arg or Trp at position 44, Met at position 45, Leu at position 65, Thr at position 66, Ser at position 67, His at position 69, and / or Val at position 73. In some embodiments, the modified CH2 domain comprises Asp at position 41, Met or Asp at position 42, Trp at position 43, Glu or Thr at position 44, Tyr or Trp at position 45, Trp at position 65, Val, Ile, or Lys at position 66, Lys, Ala, or Leu at position 67, Leu or Pro at position 69, and / or Trp at position 73.

[0034] In some embodiments, the modified CH2 domain has at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 4-113 of any one of SEQ ID NOs:86-90. In some embodiments, the modified CH2 domain has at least 85% identity to amino acids 4-113 of SEQ ID NO:1, with the proviso that the percent identity does not include the set of positions 41, 42, 43, 44, 45, 65, 66, 67, 69, and 73. In some embodiments, the modified CH2 domain comprises amino acids 41-45 and / or 65-73 of any one of SEQ ID NOs:86-90.

[0035] In another aspect, provided are polypeptides comprising a modified CH2 domain that specifically binds to a transferrin receptor, wherein the modified CH2 domain comprises 4, 5, 6, 7, 8, or 9 substitutions at the set of amino acid positions comprising: 45, 47, 49, 95, 97, 99, 102, 103, and 104, wherein the substitutions and positions are determined with reference to amino acids 4-113 of SEQ ID NO: 1. In some embodiments, the modified CH2 domain comprises a Trp at position 103. In some embodiments, the modified CH2 domain comprises at least one position selected from the following: Trp, Val, Ile, or Ala at position 45; Trp or Gly at position 47; Tyr, Arg, or Glu at position 49; Ser, Arg, or Gln at position 95; Val, Ser, or Phe at position 97; Ile, Ser, or Trp at position 99; Trp, Thr, Ser, Arg, or Asp at position 102; Trp at position 103; and Ser, Lys, Arg, or Val at position 104. In some embodiments, the modified CH2 domain comprises two, three, four, five, six, seven, eight, or nine positions selected from the following: Trp, Val, Ile, or Ala at position 45; Trp or Gly at position 47; Tyr, Arg, or Glu at position 49; Ser, Arg, or Gln at position 95; Val, Ser, or Phe at position 97; Ile, Ser, or Trp at position 99; Trp, Thr, Ser, Arg, or Asp at position 102; Trp at position 103; and Ser, Lys, Arg, or Val at position 104.

[0036] In some embodiments, the modified CH2 domain comprises Val or Ile at position 45, Gly at position 47, Arg at position 49, Arg at position 95, Ser at position 97, Ser at position 99, Thr, Ser, or Arg at position 102, Trp at position 103, and / or Lys or Arg at position 104.

[0037] In some embodiments, the modified CH2 domain has at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 4-113 of any one of SEQ ID NOs:91-95. In some embodiments, the modified CH2 domain has at least 85% identity to amino acids 4-113 of SEQ ID NO:1, with the proviso that the percent identity does not include the set of positions 45, 47, 49, 95, 97, 99, 102, 103, and 104. In some embodiments, the modified CH2 domain comprises amino acids 45-49 and / or 95-104 of any one of SEQ ID NOs:91-95.

[0038] In some embodiments, the corresponding unmodified CH2 domain is a CH2 domain of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the modified CH2 domain contains one or both of the following sets of modifications relative to the amino acid sequence of SEQ ID NO:1: (a) Ala at positions 7 and 8 (L7A and L8A), and (b) Tyr at position 25 (M25Y), Thr at position 27 (S27T), and Glu at position 29 (T29E). In some embodiments, set (a) further includes a Gly at position 102 (P102G). In some embodiments, the polypeptide is linked to a CH3 domain. In some embodiments, the CH3 domain comprises (i) a Trp at position 139 (T139W), or (ii) a Ser at position 139 (T139S), an Ala at position 141 (L141A), and a Val at position 180 (Y180V), where the amino acid positions are determined with reference to the amino acid sequence of SEQ ID NO:1. In some embodiments, the CH3 domain comprises (i) a Leu at position 201 (M201L) and a Ser at position 207 (N207S), or (ii) a Ser or an Ala at position 207 (N207S or N207A), where the amino acid positions are determined with reference to the amino acid sequence of SEQ ID NO:1. In some embodiments, the polypeptide is further linked to a Fab. In some embodiments, the Fab binds to a tau protein (e.g., a human tau protein) or a fragment thereof. The tau protein may be a phosphorylated tau protein, a non-phosphorylated tau protein, a splice isoform of tau protein, an N-terminally truncated tau protein, a C-terminally truncated tau protein, and / or a fragment thereof. In some embodiments, the Fab binds to a beta-secretase 1 (BACE1) protein (e.g., a human BACE1 protein) or a fragment thereof. The BACE1 protein may be a splice isoform of the BACE1 protein or a fragment thereof. In some embodiments, the Fab binds to a triggering receptor expressed on myeloid cells 2 (TREM2) protein (e.g., a human TREM2 protein) or a fragment thereof.In some embodiments, the Fab binds to an α-synuclein protein (e.g., a human α-synuclein protein) or a fragment thereof, which may be monomeric α-synuclein, oligomeric α-synuclein, α-synuclein fibrils, soluble α-synuclein, and / or fragments thereof.

[0039] In some embodiments, the polypeptide comprises a modified CH2 domain or a modified CH3 domain that competes for binding to the transferrin receptor with any one of the polypeptides described herein, e.g., any of SEQ ID NOs: 4-95, 236-299, 302, and 347-553. In some embodiments, the polypeptide comprises a modified CH2 domain or a modified CH3 domain that binds to the same epitope on the transferrin receptor as any one of the polypeptides described herein, e.g., any of SEQ ID NOs: 4-95, 236-299, 302, and 347-553.

[0040] In some embodiments, the polypeptide is a first polypeptide of a dimer such that the dimer is monovalent for binding to the transferrin receptor. In some embodiments, the polypeptide is a first polypeptide that binds to the transferrin receptor and forms a dimer with a second polypeptide that includes a modified CH2 domain. In some embodiments, the modified CH2 domain of the second polypeptide is the same as the modified CH2 domain of the first polypeptide.

[0041] In another aspect, there is provided a polypeptide that specifically binds to a transferrin receptor, the polypeptide comprising amino acids 157-194, or, in embodiments, amino acids 153-194 or amino acids 153-199, of any one of SEQ ID NOs: 4-29, 236-299, 422-435, amino acids 118-213 of any one of SEQ ID NOs: 30-46, amino acids 47-63 of any one of SEQ ID NOs: 47-62, amino acids 39-72 of any one of SEQ ID NOs: 63-85, amino acids 41-73 of any one of SEQ ID NOs: 86-90, or amino acids 45-104 of any one of SEQ ID NOs: 91-95.

[0042] In another aspect, provided herein is a polypeptide comprising a knob mutation and a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs:4-95, 236-299, and 422-435, wherein the knob mutation is T139W when numbered relative to SEQ ID NO:1.

[0043] In some embodiments, the polypeptide comprises a knob mutation and a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs: 349, 361, 373, 385, 397, 409, 436, 448, 460, and 472, wherein the knob mutation is T139W when numbered relative to SEQ ID NO: 1. In specific embodiments, the polypeptide comprises the sequence of any one of SEQ ID NOs: 349, 361, 373, 385, 397, 409, 436, 448, 460, and 472.

[0044] In another aspect, provided herein is a polypeptide comprising a knob mutation, an effector function-modulating mutation, and a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs:4-95, 236-299, and 422-435, wherein the knob mutation is T139W and the effector function-modulating mutation is L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1.

[0045] In some embodiments, the polypeptide comprises a knob mutation, a mutation that modulates effector function, and a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs:350, 362, 374, 386, 398, 410, 437, 449, 461, and 473, wherein the knob mutation is T139W and the mutations that modulate effector function are L7A and L8A when numbered relative to SEQ ID NO: 1. In specific embodiments, the polypeptide comprises the sequence of any one of SEQ ID NOs:350, 362, 374, 386, 398, 410, 437, 449, 461, and 473.

[0046] In some embodiments, the polypeptide comprises a knob mutation, an effector function-modulating mutation, and a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs:351, 363, 375, 387, 399, 411, 438, 450, 462, and 474, wherein the knob mutation is T139W and the effector function-modulating mutations are L7A, L8A, and P102G when numbered relative to SEQ ID NO: 1. In specific embodiments, the polypeptide comprises the sequence of any one of SEQ ID NOs:351, 363, 375, 387, 399, 411, 438, 450, 462, and 474.

[0047] In another aspect, provided herein is a polypeptide comprising a knob mutation, a serum stability-enhancing mutation, and a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs:4-95, 236-299, and 422-435, wherein the knob mutation is T139W when numbered relative to SEQ ID NO:1, and the serum stability-enhancing mutation is (i) M25Y, S27T, and T29E when numbered relative to SEQ ID NO:1, or (ii) N207S with or without M201L when numbered relative to SEQ ID NO:1.

[0048] In some embodiments, the polypeptide comprises a knob mutation, a serum stability-enhancing mutation, and a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs: 352, 364, 376, 388, 400, 412, 439, 451, 463, and 475, wherein the knob mutation is T139W and the serum stability-enhancing mutations are M25Y, S27T, and T29E, when numbered relative to SEQ ID NO: 1. In specific embodiments, the polypeptide comprises the sequence of any one of SEQ ID NOs: 352, 364, 376, 388, 400, and 412.

[0049] In some embodiments, the polypeptide comprises a knob mutation, a serum stability-enhancing mutation, and a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs:485, 492, 499, 506, 513, 520, 527, 534, 541, and 548, wherein the knob mutation is T139W and the serum stability-enhancing mutation is N207S with or without M201L when numbered relative to SEQ ID NO: 1. In specific embodiments, the polypeptide comprises the sequence of any one of SEQ ID NOs:485, 492, 499, 506, 513, 520, 527, 534, 541, and 548.

[0050] In another aspect, provided herein is a polypeptide comprising a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability; and a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs:4-95, 236-299, and 422-435, wherein the knob mutation is T139W when numbered relative to SEQ ID NO:1, the effector function mutation is L7A, L8A, and / or P102G (e.g., L7A and L8A), and the serum stability mutation is (i) M25Y, S27T, and T29E when numbered relative to SEQ ID NO:1, or (ii) N207S with or without M201L when numbered relative to SEQ ID NO:1.

[0051] In some embodiments, the polypeptide comprises a knob mutation, an effector function-modulating mutation, a serum stability-enhancing mutation, and a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs: 353, 365, 377, 389, 401, 413, 440, 452, 464, and 476, wherein the knob mutation is T139W, the effector function-modulating mutations are L7A and L8A, and the serum stability-enhancing mutations are M25Y, S27T, and T29E, when numbered relative to SEQ ID NO: 1. In a specific embodiment, the polypeptide comprises the sequence of any one of SEQ ID NOs: 353, 365, 377, 389, 401, 413, 440, 452, 464, and 476.

[0052] In some embodiments, the polypeptide comprises a knob mutation, a mutation that modulates effector function, a mutation that increases serum stability, and a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs:486, 493, 500, 507, 514, 521, 528, 535, 542, and 549, wherein, when numbered relative to SEQ ID NO: 1, the knob mutation is T139W, the mutations that modulate effector function are L7A and L8A, and the mutation that increases serum stability is N207S with or without M201L. In a specific embodiment, the polypeptide comprises the sequence of any one of SEQ ID NOs:486, 493, 500, 507, 514, 521, 528, 535, 542, and 549.

[0053] In some embodiments, the polypeptide comprises a knob mutation, an effector function-modulating mutation, a serum stability-enhancing mutation, and a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs:354, 366, 378, 390, 402, 414, 441, 453, 465, and 477, wherein, when numbered relative to SEQ ID NO: 1, the knob mutation is T139W, the effector function-modulating mutations are L7A, L8A, and P102G, and the serum stability-enhancing mutations are M25Y, S27T, and T29E. In a specific embodiment, the polypeptide comprises the sequence of any one of SEQ ID NOs:354, 366, 378, 390, 402, 414, 441, 453, 465, and 477.

[0054] In some embodiments, the polypeptide comprises a knob mutation, a mutation that modulates effector function, a mutation that increases serum stability, and a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs:487, 494, 501, 508, 515, 522, 529, 536, 543, and 550, wherein, when numbered relative to SEQ ID NO: 1, the knob mutation is T139W, the effector function mutations are L7A, L8A, and P102G, and the serum stability mutation is N207S with or without M201L. In a specific embodiment, the polypeptide comprises the sequence of any one of SEQ ID NOs:487, 494, 501, 508, 515, 522, 529, 536, 543, and 550.

[0055] In another aspect, provided herein is a polypeptide comprising a hole mutation and a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs: 4-95, 236-299, and 422-435, wherein the hole mutations are T139S, L141A, and Y180V when numbered relative to SEQ ID NO: 1.

[0056] In some embodiments, the polypeptide comprises a hole mutation and a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs: 355, 367, 379, 391, 403, 415, 442, 454, 466, and 478, wherein the hole mutations are T139S, L141A, and Y180V when numbered relative to SEQ ID NO: 1. In specific embodiments, the polypeptide comprises the sequence of any one of SEQ ID NOs: 355, 367, 379, 391, 403, 415, 442, 454, 466, and 478.

[0057] In another aspect, provided herein are polypeptides comprising a hole mutation, a mutation that modulates effector function, and a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs:4-95, 236-299, and 422-435, wherein, when numbered relative to SEQ ID NO:1, the hole mutations are T139S, L141A, and Y180V, and the mutation that modulates effector function is L7A, L8A, and / or P102G (e.g., L7A and L8A).

[0058] In some embodiments, the polypeptide comprises a hole mutation, a mutation that modulates effector function, and a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs: 356, 368, 380, 392, 404, 416, 443, 455, 467, and 479, where the hole mutations are T139S, L141A, and Y180V and the mutations that modulate effector function are L7A and L8A when numbered relative to SEQ ID NO: 1. In specific embodiments, the polypeptide comprises the sequence of any one of SEQ ID NOs: 356, 368, 380, 392, 404, 416, 443, 455, 467, and 479.

[0059] In some embodiments, the polypeptide comprises hole mutations, mutations that modulate effector function, and a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs:357, 369, 381, 393, 405, 417, 444, 456, 468, and 480, where the hole mutations are T139S, L141A, and Y180V and the effector function mutations are L7A, L8A, and P102G when numbered relative to SEQ ID NO: 1. In specific embodiments, the polypeptide comprises the sequence of any one of SEQ ID NOs:357, 369, 381, 393, 405, 417, 444, 456, 468, and 480.

[0060] In another aspect, provided herein is a polypeptide comprising a hole mutation, a mutation that increases serum stability, and a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs:4-95, 236-299, and 422-435, wherein the hole mutations are T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1, and the mutation that increases serum stability is (i) M25Y, S27T, and T29E when numbered relative to SEQ ID NO:1, or (ii) N207S with or without M201L when numbered relative to SEQ ID NO:1.

[0061] In some embodiments, the polypeptide comprises hole mutations, serum stability-enhancing mutations, and a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs:358, 370, 382, ​​394, 406, 418, 445, 457, 469, and 481, where the hole mutations are T139S, L141A, and Y180V and the serum stability-enhancing mutations are M25Y, S27T, and T29E, when numbered relative to SEQ ID NO: 1. In a specific embodiment, the polypeptide comprises the sequence of any one of SEQ ID NOs:358, 370, 382, ​​394, 406, 418, 445, 457, 469, and 481.

[0062] In some embodiments, the polypeptide comprises a hole mutation, a mutation that increases serum stability, and a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs:488, 495, 502, 509, 516, 523, 530, 537, 544, and 551, where the hole mutations are T139S, L141A, and Y180V and the serum stability-enhancing mutation is N207S with or without M201L, when numbered relative to SEQ ID NO: 1. In a specific embodiment, the polypeptide comprises the sequence of any one of SEQ ID NOs:488, 495, 502, 509, 516, 523, 530, 537, 544, and 551.

[0063] In another aspect, provided herein is a polypeptide comprising a hole mutation, a mutation that modulates effector function, and a mutation that increases serum stability, and a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs:4-95, 236-299, and 422-435, wherein the hole mutations are T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1, the effector function-modulating mutations are L7A, L8A, and / or P102G (e.g., L7A and L8A), and the serum stability-enhancing mutation is (i) M25Y, S27T, and T29E when numbered relative to SEQ ID NO:1, or (ii) N207S with or without M201L when numbered relative to SEQ ID NO:1.

[0064] In some embodiments, the polypeptide comprises a hole mutation, a mutation that modulates effector function, a mutation that increases serum stability, and a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs:303-345, 359, 371, 383, 395, 407, 419, 446, 458, 470, and 482, where the hole mutations are T139S, L141A, and Y180V, the effector function mutations are L7A and L8A, and the serum stability mutations are M25Y, S27T, and T29E, when numbered relative to SEQ ID NO: 1. In a specific embodiment, the polypeptide comprises any one of SEQ ID NOs:359, 371, 383, 395, 407, 419, 446, 458, 470, and 482.

[0065] In some embodiments, the polypeptide comprises a hole mutation, a mutation that modulates effector function, a mutation that increases serum stability, and a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs:489, 496, 503, 510, 517, 524, 531, 538, 545, and 552, where, when numbered relative to SEQ ID NO: 1, the hole mutations are T139S, L141A, and Y180V, the effector function mutations are L7A and L8A, and the serum stability mutation is N207S with or without M201L. In a specific embodiment, the polypeptide comprises the sequence of any one of SEQ ID NOs:489, 496, 503, 510, 517, 524, 531, 538, 545, and 552.

[0066] In some embodiments, the polypeptide comprises a hole mutation, a mutation that modulates effector function, a mutation that increases serum stability, and a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs: 360, 372, 384, 396, 408, 420, 447, 459, 471, and 483, where the hole mutations are T139S, L141A, and Y180V, the effector function mutations are L7A, L8A, and P102G, and the serum stability mutations are M25Y, S27T, and T29E, when numbered relative to SEQ ID NO: 1. In a specific embodiment, the polypeptide comprises the sequence of any one of SEQ ID NOs: 360, 372, 384, 396, 408, 420, 447, 459, 471, and 483.

[0067] In some embodiments, the polypeptide comprises a hole mutation, a mutation that modulates effector function, a mutation that increases serum stability, and a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs:490, 497, 504, 511, 518, 525, 532, 539, 546, and 553, where, numbered relative to SEQ ID NO: 1, the hole mutations are T139S, L141A, and Y180V, the effector function mutations are L7A, L8A, and P102G, and the serum stability mutation is N207S with or without M201L. In a specific embodiment, the polypeptide comprises the sequence of any one of SEQ ID NOs:490, 497, 504, 511, 518, 525, 532, 539, 546, and 553.

[0068] In another aspect, a polypeptide that specifically binds to a transferrin receptor is provided, comprising the sequence of any one of SEQ ID NOs: 116-233, 303-345, and 581-608.

[0069] In some embodiments, provided are polypeptides that specifically bind to a transferrin receptor, the polypeptide comprising a first sequence of any one of SEQ ID NOs: 116-130 and a second sequence independently selected from the group consisting of SEQ ID NOs: 131-139. In some embodiments, the polypeptide comprises a first sequence of any one of SEQ ID NOs: 121, 116, 122, 123, or 126-130 and a second sequence independently selected from the group consisting of SEQ ID NOs: 136, 137, and 139. In some embodiments, the polypeptide comprises a first sequence of any one of SEQ ID NOs: 120 or 124-126 and a second sequence independently selected from the group consisting of SEQ ID NOs: 135, 138, and 139.

[0070] In some embodiments, the polypeptide is selected from the group consisting of SEQ ID NO:116 and SEQ ID NO:131, SEQ ID NO:116 and SEQ ID NO:136, SEQ ID NO:117 and SEQ ID NO:132, SEQ ID NO:118 and SEQ ID NO:133, SEQ ID NO:119 and SEQ ID NO:134, SEQ ID NO:120 and SEQ ID NO:135, SEQ ID NO:121 and SEQ ID NO:136, SEQ ID NO:122 and SEQ ID NO:137, SEQ ID NO:123 and SEQ ID NO:136, SEQ ID NO:124 and SEQ ID NO:138, SEQ ID NO:125 and SEQ ID NO:135, SEQ ID NO:126 and SEQ ID NO:139, SEQ ID NO:127 and SEQ ID NO:136, SEQ ID NO:137 and SEQ ID NO:138, SEQ ID NO:139 and SEQ ID NO:139, SEQ ID NO:139 and SEQ ID NO:136, SEQ ID NO:139 and SEQ ID NO:136, SEQ ID NO:138 and SEQ ID NO:139, SEQ ID NO:139 and SEQ ID NO:136 ... SEQ ID NO:128 and SEQ ID NO:136, SEQ ID NO:129 and SEQ ID NO:136, or SEQ ID NO:130 and SEQ ID NO:136.

[0071] In some embodiments, a polypeptide is provided that specifically binds to a transferrin receptor, the polypeptide comprising a first sequence of any one of SEQ ID NOs:303-339 and a second sequence independently selected from the group consisting of SEQ ID NOs:136, 138, and 340-345. In some embodiments, the polypeptide is selected from the group consisting of SEQ ID NO:303 and SEQ ID NO:340, SEQ ID NO:304 and SEQ ID NO:340, SEQ ID NO:305 and SEQ ID NO:340, SEQ ID NO:306 and SEQ ID NO:341, SEQ ID NO:307 and SEQ ID NO:340, SEQ ID NO:308 and SEQ ID NO:340, SEQ ID NO:309 and SEQ ID NO:340, SEQ ID NO:310 and SEQ ID NO:340, SEQ ID NO:311 and SEQ ID NO:340, SEQ ID NO:312 and SEQ ID NO:341, SEQ ID NO:313 and SEQ ID NO:340, SEQ ID NO:314 and SEQ ID NO:340, SEQ ID NO:315 and SEQ ID NO:340, SEQ ID NO:316 and SEQ ID NO:341 NO:316 and SEQ ID NO:340, SEQ ID NO:317 and SEQ ID NO:340, SEQ ID NO:318 and SEQ ID NO:341, SEQ ID NO:319 and SEQ ID NO:340, SEQ ID NO:320 and SEQ ID NO:340, SEQ ID NO:321 and SEQ ID NO:340, SEQ ID NO:322 and SEQ ID NO:340, SEQ ID NO:323 and SEQ ID NO:340, SEQ ID NO:324 and SEQ ID NO:341, SEQ ID NO:325 and SEQ ID NO:340, SEQ ID NO:326 and SEQ ID NO:340, SEQ ID NO:327 and SEQ ID NO:340, SEQ ID NO:328 and SEQ ID NO:340, SEQ ID NO:329 and SEQ ID NO:340, SEQ ID NO:330 and SEQ ID NO:341, SEQ ID NO:331 and SEQ ID NO:340, SEQ ID NO:332 and SEQ IDNO:340, SEQ ID NO:306 and SEQ ID NO:340, SEQ ID NO:312 and SEQ ID NO:340, SEQ ID NO:324 and SEQ ID NO:138, SEQ ID NO:318 and SEQ ID NO:340, SEQ ID NO:324 and SEQ ID NO:340, SEQ ID NO:330 and SEQ ID NO:340, SEQ ID NO:318 and SEQ ID NO:138, SEQ ID NO:333 and SEQ ID NO:136, SEQ ID NO:334 and SEQ ID NO:136, SEQ ID NO:312 and SEQ ID NO:138, SEQ ID NO:333 and SEQ ID NO:342, SEQ ID NO:335 and SEQ ID NO:342, SEQ ID NO:336 and SEQ ID NO:342, SEQ ID SEQ ID NO:334 and SEQ ID NO:342, SEQ ID NO:330 and SEQ ID NO:138, SEQ ID NO:330 and SEQ ID NO:343, SEQ ID NO:330 and SEQ ID NO:345, SEQ ID NO:337 and SEQ ID NO:136, SEQ ID NO:338 and SEQ ID NO:136, SEQ ID NO:339 and SEQ ID NO:136, SEQ ID NO:330 and SEQ ID NO:344, SEQ ID NO:312 and SEQ ID NO:343, or SEQ ID NO:312 and SEQ ID NO:345.

[0072] In another aspect, the disclosure features a polypeptide that specifically binds to a transferrin receptor, the polypeptide comprising a first sequence of any one of SEQ ID NOs:581, 583, 585, 587, 589, 591, and 593, and a second sequence independently selected from the group consisting of SEQ ID NOs:582, 584, 586, 588, 590, 592, and 594. In specific embodiments, the polypeptide comprises SEQ ID NO:581 and SEQ ID NO:582, SEQ ID NO:583 and SEQ ID NO:584, SEQ ID NO:585 and SEQ ID NO:586, SEQ ID NO:587 and SEQ ID NO:588, SEQ ID NO:589 and SEQ ID NO:590, SEQ ID NO:591 and SEQ ID NO:592, or SEQ ID NO:593 and SEQ ID NO:594.

[0073] In another aspect, the disclosure features a polypeptide that specifically binds to a transferrin receptor, the polypeptide comprising a first sequence of any one of SEQ ID NOs:554, 557, and 560, and a second sequence independently selected from the group consisting of SEQ ID NOs:555, 558, and 561. In specific embodiments, the polypeptide comprises SEQ ID NOs:554 and SEQ ID NOs:555, SEQ ID NOs:557 and SEQ ID NOs:558, or SEQ ID NOs:560 and SEQ ID NOs:561.

[0074] In another aspect, the disclosure features a polypeptide that specifically binds to a transferrin receptor, the polypeptide including a first sequence of any one of SEQ ID NOs:554, 557, and 560, and a second sequence independently selected from the group consisting of SEQ ID NOs:340-345, 582, 584, 586, 588, 590, 592, and 594. In specific embodiments, the polypeptide is selected from the group consisting of SEQ ID NO:554 and SEQ ID NO:340, SEQ ID NO:554 and SEQ ID NO:341, SEQ ID NO:554 and SEQ ID NO:342, SEQ ID NO:554 and SEQ ID NO:343, SEQ ID NO:554 and SEQ ID NO:344, SEQ ID NO:554 and SEQ ID NO:345, SEQ ID NO:554 and SEQ ID NO:582, SEQ ID NO:554 and SEQ ID NO:584, SEQ ID NO:554 and SEQ ID NO:586, SEQ ID NO:554 and SEQ ID NO:588, SEQ ID NO:554 and SEQ ID NO:590, SEQ ID NO:554 and SEQ ID NO:592, SEQ ID NO:554 and SEQ ID NO:594, SEQ ID NO:557 and SEQ ID NO:340, SEQ ID NO:557 and SEQ ID NO:341, SEQ ID NO:557 and SEQ ID NO:342, SEQ ID NO:557 and SEQ ID NO:343, SEQ ID NO:557 and SEQ ID NO:344, SEQ ID NO:557 and SEQ ID NO:345, SEQ ID NO:557 and SEQ ID NO:582, SEQ ID NO:557 and SEQ ID NO:584, SEQ ID NO:557 and SEQ ID NO:586, SEQ ID NO:557 and SEQ ID NO:588, SEQ ID NO:557 and SEQ ID NO:590, SEQ ID NO:557 and SEQ ID NO:592, SEQ ID NO:557 and SEQ ID NO:594, SEQ ID NO:560 and SEQ ID NO:340, SEQ ID NO:560 and SEQ ID NO:341, SEQ ID NO:560 and SEQ ID NO:342, SEQ IDSEQ ID NO:560 and SEQ ID NO:343, SEQ ID NO:560 and SEQ ID NO:344, or SEQ ID NO:560 and SEQ ID NO:345, SEQ ID NO:560 and SEQ ID NO:582, SEQ ID NO:560 and SEQ ID NO:584, SEQ ID NO:560 and SEQ ID NO:586, SEQ ID NO:560 and SEQ ID NO:588, SEQ ID NO:560 and SEQ ID NO:590, SEQ ID NO:560 and SEQ ID NO:592, or SEQ ID NO:560 and SEQ ID NO:594.

[0075] In another aspect, the disclosure features a polypeptide that specifically binds to a transferrin receptor, the polypeptide including a first sequence of any one of SEQ ID NOs:303-339, 581, 583, 585, 587, 589, 591, and 593, and a second sequence independently selected from the group consisting of SEQ ID NOs:555, 558, and 561. In specific embodiments, the polypeptide is selected from the group consisting of SEQ ID NO:303 and SEQ ID NO:555, SEQ ID NO:303 and SEQ ID NO:558, SEQ ID NO:303 and SEQ ID NO:561, SEQ ID NO:304 and SEQ ID NO:555, SEQ ID NO:304 and SEQ ID NO:558, SEQ ID NO:304 and SEQ ID NO:561, SEQ ID NO:305 and SEQ ID NO:555, SEQ ID NO:305 and SEQ ID NO:558, SEQ ID NO:305 and SEQ ID NO:561, SEQ ID NO:306 and SEQ ID NO:555, SEQ ID NO:306 and SEQ ID NO:558, SEQ ID NO:306 and SEQ ID NO:561, SEQ ID NO:307 and SEQ ID NO:555, SEQ ID NO:307 and SEQ ID NO:558, SEQ ID NO:307 and SEQ ID NO:561, SEQ ID NO:308 and SEQ ID NO:555, SEQ ID NO:308 and SEQ ID NO:558, SEQ ID NO:308 and SEQ ID NO:561, SEQ ID NO:309 and SEQ ID NO:555, SEQ ID NO:309 and SEQ ID NO:558, SEQ ID NO:309 and SEQ ID NO:561, SEQ ID NO:310 and SEQ ID NO:555, SEQ ID NO:310 and SEQ ID NO:558, SEQ ID NO:310 and SEQ ID NO:561, SEQ ID NO:311 and SEQ ID NO:555, SEQ ID NO:311 and SEQ ID NO:558, SEQ ID NO:311 and SEQ ID NO:561, SEQ ID NO:312 and SEQ ID NO:555, SEQ ID NO:312 and SEQ ID NO:558, SEQ IDNO:312 and SEQ ID NO:561, SEQ ID NO:313 and SEQ ID NO:555, SEQ ID NO:313 and SEQ ID NO:558, SEQ ID NO:313 and SEQ ID NO:561, SEQ ID NO:314 and SEQ ID NO:555, SEQ ID NO:314 and SEQ ID NO:558, SEQ ID NO:314 and SEQ ID NO:561, SEQ ID NO:315 and SEQ ID NO:555, SEQ ID NO:315 and SEQ ID NO:558, SEQ ID NO:315 and SEQ ID NO:561, SEQ ID NO:316 and SEQ ID NO:555, SEQ ID NO:316 and SEQ ID NO:558, SEQ ID NO:316 and SEQ ID NO:561, SEQ ID NO:317 and SEQ ID NO:555, SEQ ID NO:317 and SEQ ID NO:558, SEQ ID NO:317 and SEQ ID NO:561, SEQ ID NO:318 and SEQ ID NO:555, SEQ ID NO:318 and SEQ ID NO:558, SEQ ID NO:318 and SEQ ID NO:561, SEQ ID NO:319 and SEQ ID NO:555, SEQ ID NO:319 and SEQ ID NO:558, SEQ ID NO:319 and SEQ ID NO:561, SEQ ID NO:320 and SEQ ID NO:555, SEQ ID NO:320 and SEQ ID NO:558, SEQ ID NO:320 and SEQ ID NO:561, SEQ ID NO:321 and SEQ ID NO:555, SEQ ID NO:321 and SEQ ID NO:558, SEQ ID NO:321 and SEQ ID NO:561, SEQ ID NO:322 and SEQ ID NO:555, SEQ ID NO:322 and SEQ ID NO:558, SEQ ID NO:322 and SEQ ID NO:561, SEQ ID NO:323 and SEQ ID NO:555, SEQ ID NO:323 and SEQ ID NO:558, SEQ ID NO:323 and SEQ ID NO:561, SEQ ID NO:324 and SEQ ID NO:555, SEQ ID NO:324 and SEQ IDNO:558, SEQ ID NO:324 and SEQ ID NO:561, SEQ ID NO:325 and SEQ ID NO:555, SEQ ID NO:325 and SEQ ID NO:558, SEQ ID NO:325 and SEQ ID NO:561, SEQ ID NO:326 and SEQ ID NO:555, SEQ ID NO:326 and SEQ ID NO:558, SEQ ID NO:326 and SEQ ID NO:561, SEQ ID NO:327 and SEQ ID NO:555, SEQ ID NO:327 and SEQ ID NO:558, SEQ ID NO:327 and SEQ ID NO:561, SEQ ID NO:328 and SEQ ID NO:555, SEQ ID NO:328 and SEQ ID NO:558, SEQ ID NO:328 and SEQ ID NO:561, SEQ ID NO:329 and SEQ ID NO:555, SEQ ID NO:329 and SEQ ID NO:558, SEQ ID NO:329 and SEQ ID NO:561, SEQ ID NO:330 and SEQ ID NO:555, SEQ ID NO:330 and SEQ ID NO:558, SEQ ID NO:330 and SEQ ID NO:561, SEQ ID NO:331 and SEQ ID NO:555, SEQ ID NO:331 and SEQ ID NO:558, SEQ ID NO:331 and SEQ ID NO:561, SEQ ID NO:332 and SEQ ID NO:555, SEQ ID NO:332 and SEQ ID NO:558, SEQ ID NO:332 and SEQ ID NO:561, SEQ ID NO:333 and SEQ ID NO:555, SEQ ID NO:333 and SEQ ID NO:558, SEQ ID NO:333 and SEQ ID NO:561, SEQ ID NO:334 and SEQ ID NO:555, SEQ ID NO:334 and SEQ ID NO:558, SEQ ID NO:334 and SEQ ID NO:561, SEQ ID NO:335 and SEQ ID NO:555, SEQ ID NO:335 and SEQ ID NO:558, SEQ ID NO:335 and SEQ ID NO:561, SEQ ID NO:336 and SEQ ID NO:555, SEQ IDNO:336 and SEQ ID NO:558, SEQ ID NO:336 and SEQ ID NO:561, SEQ ID NO:337 and SEQ ID NO:555, SEQ ID NO:337 and SEQ ID NO:558, SEQ ID NO:337 and SEQ ID NO:561, SEQ ID NO:338 and SEQ ID NO:555, SEQ ID NO:338 and SEQ ID NO:558, SEQ ID NO:338 and SEQ ID NO:561, SEQ ID NO:339 and SEQ ID NO:555, SEQ ID NO:339 and SEQ ID NO:558, SEQ ID NO:339 and SEQ ID NO:561, SEQ ID NO:581 and SEQ ID NO:555, SEQ ID NO:581 and SEQ ID NO:558, SEQ ID NO:581 and SEQ ID NO:561, SEQ ID NO:583 and SEQ ID NO:555, SEQ ID NO:583 and SEQ ID NO:558, SEQ ID NO:583 and SEQ ID NO:561, SEQ ID NO:585 and SEQ ID NO:555, SEQ ID NO:585 and SEQ ID NO:558, SEQ ID NO:585 and SEQ ID NO:561, SEQ ID NO:587 and SEQ ID NO:555, SEQ ID NO:587 and SEQ ID NO:558, SEQ ID NO:587 and SEQ ID NO:561, SEQ ID NO:589 and SEQ ID NO:555, SEQ ID NO:589 and SEQ ID NO:558, SEQ ID NO:589 and SEQ ID NO:561, SEQ ID NO:591 and SEQ ID NO:555, SEQ ID SEQ ID NO:591 and SEQ ID NO:558, SEQ ID NO:591 and SEQ ID NO:561, SEQ ID NO:593 and SEQ ID NO:555, SEQ ID NO:593 and SEQ ID NO:558, or SEQ ID NO:593 and SEQ ID NO:561.

[0076] In another aspect, the disclosure features a polypeptide that specifically binds to a transferrin receptor, the polypeptide including a first sequence of any one of SEQ ID NOs:609-614 and a second sequence independently selected from the group consisting of SEQ ID NOs:615-620.

[0077] In another aspect, the disclosure features a polypeptide that specifically binds to a transferrin receptor, the polypeptide including a first sequence of any one of SEQ ID NOs:303, 312, 315-318, and 328, and a second sequence independently selected from the group consisting of SEQ ID NOs:135, 340, and 341. In specific embodiments, the polypeptide comprises SEQ ID NO:303 and SEQ ID NO:340, SEQ ID NO:316 and SEQ ID NO:340, SEQ ID NO:317 and SEQ ID NO:340, SEQ ID NO:318 and SEQ ID NO:340, SEQ ID NO:328 and SEQ ID NO:341, SEQ ID NO:318 and SEQ ID NO:340, SEQ ID NO:312 and SEQ ID NO:340, SEQ ID NO:303 and SEQ ID NO:341, SEQ ID NO:316 and SEQ ID NO:135, or SEQ ID NO:315 and SEQ ID NO:341.

[0078] In another aspect, the disclosure features a polypeptide that specifically binds to a transferrin receptor, the polypeptide comprising a first sequence of any one of SEQ ID NOs:595, 597, 599, 601, 603, 605, and 607, and a second sequence independently selected from the group consisting of SEQ ID NOs:596, 598, 600, 602, 604, 606, and 608. In specific embodiments, the polypeptide comprises SEQ ID NO:595 and SEQ ID NO:596, SEQ ID NO:597 and SEQ ID NO:598, SEQ ID NO:599 and SEQ ID NO:600, SEQ ID NO:601 and SEQ ID NO:602, SEQ ID NO:603 and SEQ ID NO:604, SEQ ID NO:605 and SEQ ID NO:606, or SEQ ID NO:607 and SEQ ID NO:608.

[0079] In another aspect, the disclosure features a polypeptide that specifically binds to a transferrin receptor, the polypeptide comprising a first sequence of either of SEQ ID NOs:575 and 578 and a second sequence independently selected from the group consisting of SEQ ID NOs:576 and 579. In specific embodiments, the polypeptide comprises SEQ ID NO:575 and SEQ ID NO:576, or SEQ ID NO:578 and SEQ ID NO:579.

[0080] In another aspect, the disclosure relates to a polypeptide that specifically binds to a transferrin receptor, comprising a first sequence of any one of SEQ ID NOs:575 and 578 and a second sequence independently selected from the group consisting of SEQ ID NOs:136, 138, and 340-345.

[0081] In another aspect, the disclosure relates to a polypeptide that specifically binds to a transferrin receptor, comprising a first sequence of any one of SEQ ID NOs:303-339 and a second sequence independently selected from the group consisting of SEQ ID NOs:576 and 579.

[0082] In some embodiments, a polypeptide is provided that specifically binds to a transferrin receptor, the polypeptide comprising a first sequence of any one of SEQ ID NOs:140-153 and a second sequence independently selected from the group consisting of SEQ ID NOs:154-157. In some embodiments, the polypeptide is selected from the group consisting of SEQ ID NO:140 and SEQ ID NO:154, SEQ ID NO:141 and SEQ ID NO:154, SEQ ID NO:142 and SEQ ID NO:154, SEQ ID NO:143 and SEQ ID NO:154, SEQ ID NO:144 and SEQ ID NO:154, SEQ ID NO:145 and SEQ ID NO:154, SEQ ID NO:146 and SEQ ID NO:154, SEQ ID NO:147 and SEQ ID NO:154, SEQ ID NO:148 and SEQ ID NO:155, SEQ ID NO:149 and SEQ ID NO:154, SEQ ID NO:140 and SEQ ID NO:156, SEQ ID NO:150 and SEQ ID NO:156, SEQ ID NO:151 and SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID SEQ ID NO:152 and SEQ ID NO:155, or SEQ ID NO:153 and SEQ ID NO:154.

[0083] In some embodiments, a polypeptide that specifically binds to a transferrin receptor is provided, comprising a first sequence of any one of SEQ ID NOs:158-171 and a second sequence independently selected from the group consisting of SEQ ID NOs:172-186. In some embodiments, the polypeptide is selected from the group consisting of SEQ ID NO:158 and SEQ ID NO:172, SEQ ID NO:158 and SEQ ID NO:179, SEQ ID NO:159 and SEQ ID NO:173, SEQ ID NO:159 and SEQ ID NO:181, SEQ ID NO:160 and SEQ ID NO:174, SEQ ID NO:161 and SEQ ID NO:175, SEQ ID NO:162 and SEQ ID NO:176, SEQ ID NO:163 and SEQ ID NO:177, SEQ ID NO:164 and SEQ ID NO:178, SEQ ID NO:165 and SEQ ID NO:180, SEQ ID NO:166 and SEQ ID NO:182, SEQ ID NO:167 and SEQ ID NO:183, SEQ ID NO:168 and SEQ ID NO:184, SEQ ID NO:165 SEQ ID NO:169 and SEQ ID NO:185, SEQ ID NO:170 and SEQ ID NO:174, or SEQ ID NO:171 and SEQ ID NO:186.

[0084] In some embodiments, a polypeptide that specifically binds to a transferrin receptor is provided, comprising a first sequence of any one of SEQ ID NOs:187-204 and a second sequence independently selected from the group consisting of SEQ ID NOs:205-215. In some embodiments, the polypeptide is selected from the group consisting of SEQ ID NO:187 and SEQ ID NO:205, SEQ ID NO:187 and SEQ ID NO:206, SEQ ID NO:188 and SEQ ID NO:206, SEQ ID NO:189 and SEQ ID NO:207, SEQ ID NO:190 and SEQ ID NO:206, SEQ ID NO:191 and SEQ ID NO:205, SEQ ID NO:192 and SEQ ID NO:206, SEQ ID NO:193 and SEQ ID NO:208, SEQ ID NO:194 and SEQ ID NO:206, SEQ ID NO:195 and SEQ ID NO:209, SEQ ID NO:196 and SEQ ID NO:206, SEQ ID NO:197 and SEQ ID NO:205, SEQ ID NO:198 and SEQ ID NO:206, SEQ ID SEQ ID NO:199 and SEQ ID NO:208, SEQ ID NO:200 and SEQ ID NO:206, SEQ ID NO:201 and SEQ ID NO:210, SEQ ID NO:201 and SEQ ID NO:211, SEQ ID NO:201 and SEQ ID NO:212, SEQ ID NO:202 and SEQ ID NO:212, SEQ ID NO:203 and SEQ ID NO:213, SEQ ID NO:203 and SEQ ID NO:214, or SEQ ID NO:204 and SEQ ID NO:215.

[0085] In some embodiments, a polypeptide is provided that specifically binds to a transferrin receptor, comprising a first sequence of any one of SEQ ID NOs:216-220 and a second sequence independently selected from the group consisting of SEQ ID NOs:221-224. In some embodiments, the polypeptide comprises SEQ ID NO:216 and SEQ ID NO:221, SEQ ID NO:217 and SEQ ID NO:221, SEQ ID NO:218 and SEQ ID NO:222, SEQ ID NO:219 and SEQ ID NO:223, or SEQ ID NO:220 and SEQ ID NO:224.

[0086] In some embodiments, a polypeptide is provided that specifically binds to a transferrin receptor, comprising a first sequence of any one of SEQ ID NOs:225-228 and a second sequence independently selected from the group consisting of SEQ ID NOs:229-233. In some embodiments, the polypeptide comprises SEQ ID NOs:225 and 229, SEQ ID NOs:226 and SEQ ID NO:230, SEQ ID NOs:226 and SEQ ID NO:231, SEQ ID NOs:227 and SEQ ID NO:232, or SEQ ID NOs:228 and SEQ ID NO:233.

[0087] In any of the above embodiments, the polypeptide can further include Leu to Ala substitutions (L7A and L8A) at positions 7 and 8. In some embodiments, the Ala substitutions at positions 7 and 8 (L7A and L8A) are combined with a Pro to Gly substitution at position 102 (P102G).

[0088] In yet another aspect, there is provided a polynucleotide comprising a nucleic acid sequence encoding a polypeptide that specifically binds to the transferrin receptor described herein.

[0089] In another aspect, a vector is provided that comprises a polynucleotide that includes a nucleic acid sequence encoding a polypeptide that specifically binds to a transferrin receptor described herein.

[0090] In yet another aspect, a host cell is provided that comprises a polynucleotide comprising a nucleic acid sequence encoding a polypeptide that specifically binds to a transferrin receptor described herein.

[0091] In yet another aspect, there is provided a method for producing a polypeptide comprising a modified CH3 domain or a modified CH2 domain, the method comprising culturing a host cell under conditions in which a polypeptide encoded by a polynucleotide described herein is expressed.

[0092] In another aspect, there is provided a pharmaceutical composition comprising a polypeptide that specifically binds to the transferrin receptor described herein and a pharmaceutically acceptable carrier.

[0093] In yet another aspect, methods are provided for transcytosing a composition across an endothelium, the method comprising contacting the endothelium with a composition comprising a polypeptide that specifically binds to a transferrin receptor as described herein. In some embodiments, the endothelium is the blood-brain barrier (BBB).

[0094] In yet another aspect, a method for engineering a CH3 domain to specifically bind to a transferrin receptor, comprising: (a) a polynucleotide encoding a CH3 domain, (i) 157, 159, 160, 161, 162, 163, 186, 189, and 194, or (ii) 118, 119, 120, 122, 210, 211, 212, and 213 and modifying the polypeptide to have at least five amino acid substitutions at a set of amino acid positions comprising: (b) expressing a polypeptide comprising a modified CH3 domain; (c) determining whether the modified CH3 domain binds to the transferrin receptor; A method is provided that includes:

[0095] In another aspect, a method is provided for engineering a CH3 domain to specifically bind to a transferrin receptor, the method comprising: (a) modifying a polynucleotide encoding the CH3 domain to include at least five amino acid substitutions in the CH3 domain as described in any one of the above paragraphs describing substitutions in the CH3 domain; and (b) expressing and recovering a polypeptide comprising the modified CH3 domain.

[0096] In some embodiments, the steps of expressing a polypeptide comprising a modified CH3 domain and determining whether the modified CH3 domain binds to a transferrin receptor are carried out using a display system. In some embodiments, the display system is a cell surface display system, a viral display system, an mRNA display system, a polysome display system, or a ribosome display system. In some embodiments, the polypeptide comprising a modified CH3 domain is expressed as a soluble protein.

[0097] In another embodiment, there is provided a method for engineering a CH2 domain to specifically bind to a transferrin receptor, comprising: (a) a polynucleotide encoding a CH2 domain, (i) 47, 49, 56, 58, 59, 60, 61, 62, and 63; (ii) 39, 40, 41, 42, 43, 44, 68, 70, 71, and 72; (iii) 41, 42, 43, 44, 45, 65, 66, 67, 69, and 73, or (iv) 45, 47, 49, 95, 97, 99, 102, 103, and 104 and modifying the polypeptide to have at least five amino acid substitutions at a set of amino acid positions comprising: (b) expressing a polypeptide comprising a modified CH2 domain; (c) determining whether the modified CH2 domain binds to the transferrin receptor; A method is provided that includes:

[0098] In another aspect, a method is provided for engineering a CH2 domain to specifically bind to a transferrin receptor, the method comprising: (a) modifying a polynucleotide encoding the CH2 domain to have at least five amino acid substitutions in the CH2 domain as described in any one of the above paragraphs describing substitutions in the CH2 domain; and (b) expressing and recovering a polypeptide comprising the modified CH2 domain.

[0099] In some embodiments, the steps of expressing a polypeptide comprising a modified CH2 domain and determining whether the modified CH2 domain binds to a transferrin receptor are carried out using a display system. In some embodiments, the display system is a cell surface display system, a viral display system, an mRNA display system, a polysome display system, or a ribosome display system. In some embodiments, the polypeptide comprising a modified CH2 domain is expressed as a soluble protein.

[0100] In another aspect, the disclosure features a method for increasing binding of a modified Fc polypeptide comprising a non-native binding site to a target (e.g., a transferrin receptor), the method including: (a) introducing one or more substitutions at one or more positions within 10 Å (e.g., within 9 Å, 8 Å, 7 Å, 6 Å, 5 Å, 4 Å, 3 Å, 2 Å, 1 Å) of the non-native site; and (b) testing the modified Fc polypeptide for binding to the target.

[0101] In some embodiments, the non-native binding site comprises a substitution at one or more of the following positions: 157, 159, 160, 161, 162, 163, 186, 189, and 194.

[0102] In some embodiments, one or more substitutions at one or more positions within 10 Å of the non-native binding site are selected from the group consisting of SEQ ID NO: Based on NO:1, it is selected from the group consisting of K21, R28, Q115, R117, E118, Q120, T132, K133, N134, Q135, S137, K143, E153, E155, S156, G158, Y164, K165, T166, D172, S173, D174, S176, K182, L183, T184, V185, K187, S188, Q191, Q192, G193, V195, F196, S197, S199, Q211, S213, S215, L216, S217, P218, G219, and K220. [The present invention 1001] 1. A polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor, wherein the modified CH3 domain comprises 5, 6, 7, 8, or 9 substitutions at a set of amino acid positions including positions 157, 159, 160, 161, 162, 163, 186, 189, and 194, and the substitutions and positions are determined based on amino acids 114 to 220 of SEQ ID NO:1. [The present invention 1002] 1001. The polypeptide of the present invention, wherein said modified CH3 domain further comprises one, two, three, or four substitutions at positions 153, 164, 165, and 188, inclusive. [The present invention 1003] The polypeptide of claim 1001 or 1002, wherein said modified CH3 domain further comprises one, two, or three substitutions at positions 187, 197, and 199, inclusive. [The present invention 1004] The polypeptide of any one of claims 1001 to 1003, which binds to the apical domain of the transferrin receptor. [The present invention 1005] 1004. The polypeptide of the present invention, which binds to the transferrin receptor without inhibiting the binding of transferrin to the transferrin receptor. [The present invention 1006] The polypeptide of the present invention 1004 or 1005, which binds to an epitope comprising amino acid 208 of the transferrin receptor sequence. [The present invention 1007] 1007. The polypeptide of any one of claims 1001 to 1006, wherein the modified CH3 domain comprises Trp at position 161. [The present invention 1008] The polypeptide of any of claims 1001 to 1007, wherein the modified CH3 domain comprises an aromatic amino acid at position 194. [The present invention 1009] The polypeptide of claim 1008, wherein the aromatic amino acid at position 194 is Trp or Phe. [The present invention 1010] 10. The modified CH3 domain comprising: Position 157 is Leu, Tyr, Met, or Val; position 159 is Leu, Thr, His, or Pro; position 160 is Val, Pro, or an acidic amino acid; position 161 is Trp; position 162 is Val, Ser, or Ala; position 186 is Glu, Ala, Ser, Leu, Thr, or Pro; position 189 is Thr or an acidic amino acid; and position 194 is Trp, Tyr, His, or Phe. The polypeptide of any one of claims 1001 to 1006, comprising at least one position selected from the group consisting of: [The present invention 1011] 10. The modified CH3 domain comprising: Position 157 is Leu, Tyr, Met, or Val; position 159 is Leu, Thr, His, or Pro; position 160 is Val, Pro, or an acidic amino acid; position 161 is Trp; position 162 is Val, Ser, or Ala; position 186 is Glu, Ala, Ser, Leu, Thr, or Pro; position 189 is Thr or an acidic amino acid; and position 194 is Trp, Tyr, His, or Phe. 10. The polypeptide of claim 10, comprising two, three, four, five, six, seven, or eight positions selected from: [The present invention 1012] 10. The polypeptide of any one of claims 1001 to 1011, wherein the modified CH3 domain comprises Leu or Met at position 157, Leu, His, or Pro at position 159, Val at position 160, Trp at position 161, Val or Ala at position 162, Pro at position 186, Thr at position 189, and / or Trp at position 194. [The present invention 1013] 1012. The polypeptide of the present invention, wherein said modified CH3 domain further comprises Ser, Thr, Gln, or Phe at position 164. [The present invention 1014] The polypeptide of claim 1012 or 1013, wherein the modified CH3 domain further comprises Trp, Tyr, Leu, or Gln at position 153. [The present invention 1015] The polypeptide of any one of claims 1012 to 1014, wherein the modified CH3 domain further comprises Gln, Phe, or His at position 165. [The present invention 1016] The polypeptide of claim 1012 or 1013, wherein the modified CH3 domain further comprises Trp at position 153 and / or Gln at position 165. [The present invention 1017] 10. The modified CH3 domain comprising: Lys, Arg, Gly, or Pro at position 187, Ser, Thr, Glu, or Lys at position 197, and Ser, Trp, or Gly at position 199 The polypeptide of any of claims 1010 to 1016, further comprising one, two, or three positions selected from: [The present invention 1018] The polypeptide of any of claims 1001 to 1011, wherein the modified CH3 domain comprises Tyr at position 157, Thr at position 159, Glu or Val at position 160, Trp at position 161, Ser at position 162, Ser or Thr at position 186, Glu at position 189, and / or Phe at position 194. [The present invention 1019] 1018. The polypeptide of the present invention, wherein the modified CH3 domain further comprises Trp, Tyr, Leu, or Gln at position 153. [The present invention 1020] The polypeptide of claim 1018 or 1019, wherein said modified CH3 domain further comprises Gln at position 188. [The present invention 1021] 1018. The polypeptide of the invention, wherein said modified CH3 domain further comprises Trp at position 153 and / or Gln at position 188. [The present invention 1022] The polypeptide of any one of claims 1018 to 1021, wherein the modified CH3 domain comprises Asn at position 163. [The present invention 1023] The modified CH3 domain comprises the following substitutions: Tyr at position 153, Thr at position 159, Trp at position 161, Val at position 162, Ser or Thr at position 186, Glu at position 188, and / or Phe at position 194 Any of the polypeptides of 1002 to 1006 of the present invention, comprising one or more of the following: [The present invention 1024] Any of the polypeptides of claims 1001 to 1023, wherein the modified CH3 domain has at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 114 to 220 of any one of SEQ ID NOs: 4 to 29, 236 to 299, and 422 to 435. [The present invention 1025] Any of the polypeptides of claims 1001 to 1023, wherein the modified CH3 domain has at least 85% identity with amino acids 114 to 220 of SEQ ID NO: 1, with the proviso that the set of positions 157, 159, 160, 161, 162, 163, 186, 189, and 194 are not included in the percent identity. [The present invention 1026] 1001-1025. The polypeptide of any one of claims 1001-1025, wherein the modified CH3 domain comprises amino acids 157-163 and / or 186-194 of any one of SEQ ID NOs: 4-29, 236-299, and 422-435. [The present invention 1027] 10. The modified CH3 domain comprising: Position 153 is Trp, Leu, or Glu, position 157 is Tyr or Phe, position 159 is Thr, position 160 is Glu, position 161 is Trp, position 162 is Ser, Ala, Val, or Asn, position 163 is Ser or Asn, position 186 is Thr or Ser, position 188 is Glu or Ser, position 189 is Glu, and position 194 is Phe The polypeptide of any one of 1002 to 1006, comprising at least one position selected from the group consisting of: [The present invention 1028] 10. The modified CH3 domain comprising: Position 153 is Trp, Leu, or Glu, position 157 is Tyr or Phe, position 159 is Thr, position 160 is Glu, position 161 is Trp, position 162 is Ser, Ala, Val, or Asn, position 163 is Ser or Asn, position 186 is Thr or Ser, position 188 is Glu or Ser, position 189 is Glu, and position 194 is Phe 1027. The polypeptide of the present invention, comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 positions selected from: [The present invention 1029] The modified CH3 domain has the following 11 positions: Position 153 is Trp, Leu, or Glu, position 157 is Tyr or Phe, position 159 is Thr, position 160 is Glu, position 161 is Trp, position 162 is Ser, Ala, Val, or Asn, position 163 is Ser or Asn, position 186 is Thr or Ser, position 188 is Glu or Ser, position 189 is Glu, and position 194 is Phe 1028. The polypeptide of the present invention, comprising: [The present invention 1030] The polypeptide of the invention 1028 or 1029, wherein the modified CH3 domain has at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 114-220 of any one of SEQ ID NOs: 4-29, 236-299, and 422-435. [The present invention 1031] A polypeptide of the present invention 1030, wherein at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the residues corresponding to positions 153, 157, 159, 160, 161, 162, 163, 164, 165, 186, 187, 188, 189, 194, 197, and 199 of any one of SEQ ID NOs: 4 to 29, 236 to 299, and 422 to 435 are not deleted or substituted. [The present invention 1032] Any of the polypeptides of the present invention 1001 to 1031, wherein the modified CH3 domain further comprises (i) Trp at position 139, or (ii) Ser at position 139, Ala at position 141, and Val at position 180, and the amino acid positions are determined based on SEQ ID NO:1. [The present invention 1033] Any of the polypeptides of the present invention 1001 to 1032, wherein the modified CH3 domain further comprises (i) Leu at position 201 and Ser at position 207, or (ii) Ser or Ala at position 207, and the amino acid positions are determined based on SEQ ID NO:1. [The present invention 1034] 1. A polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor, wherein the modified CH3 domain comprises one or more substitutions at a set of amino acid positions including: 153, 157, 159, 160, 162, 163, 186, 188, 189, 194, 197, and 199, wherein the substitutions and the positions are determined with reference to the sequence of SEQ ID NO:13. [This invention 1035] 1034. The polypeptide of the invention, wherein the modified CH3 domain comprises Glu, Leu, Ser, Val, Trp, or Tyr at position 153, an aromatic amino acid, Met, Pro, or Val at position 157, Thr, Asn, or Val at position 159, Glu, Ile, Pro, or Val at position 160, an aliphatic amino acid, Ser, or Thr at position 162, Ser, Asn, Arg, or Thr at position 163, Thr, His, or Ser at position 186, Glu, Ser, Asp, Gly, Thr, Pro, Gln, or Arg at position 188, Glu or Arg at position 189, Phe, His, Lys, Tyr, or Trp at position 194, Ser, Thr, or Trp at position 197, and Ser, Cys, Pro, Met, or Trp at position 199. [The present invention 1036] The polypeptide of the present invention 1035, wherein the aromatic amino acid at position 157 is Tyr, Phe, or Trp, and the aliphatic amino acid at position 162 is Ala, Ile, or Val. [This invention 1037] The polypeptide of the present invention 1034 or 1035, wherein the modified CH3 domain comprises Glu, Leu, or Trp at position 153, an aromatic amino acid at position 157, Thr at position 159, Glu at position 160, an aliphatic amino acid or Ser at position 162, Ser or Asn at position 163, Thr or Ser at position 186, Glu or Ser at position 188, Glu at position 189, Phe, His, Tyr, or Trp at position 194, Ser at position 197, and Ser at position 199. [The present invention 1038] The polypeptide of the present invention 1037, wherein the aromatic amino acid at position 157 is Tyr or Phe, and the aliphatic amino acid at position 162 is Ala or Val. [This invention 1039] The polypeptide of any one of claims 1034 to 1038, wherein the modified CH3 domain has the sequence of SEQ ID NO: 556 or 559. [The present invention 1040] Any of the polypeptides of 1034 to 1038 of the present invention, wherein the modified CH3 domain comprises one substitution at a set of amino acid positions including positions 153, 157, 159, 160, 162, 163, 186, 188, 189, 194, 197, and 199. [The present invention 1041] The polypeptide of the present invention 1040, wherein the modified CH3 domain has the sequence of any one of SEQ ID NOs: 563 to 574. [The present invention 1042] The polypeptide of the present invention 1034 or 1035, wherein the modified CH3 domain comprises Glu, Leu, or Trp at position 153, Tyr or Phe at position 157, Thr at position 159, Glu at position 160, Ala, Val, or Ser at position 162, Ser or Asn at position 163, Thr or Ser at position 186, Glu or Ser at position 188, Glu at position 189, Phe at position 194, Ser at position 197, and Ser at position 199. [This invention 1043] The polypeptide of the present invention 1042, wherein said modified CH3 domain has the sequence of SEQ ID NO:562. [This invention 1044] 1. A polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor, wherein the modified CH3 domain comprises one or more substitutions at a set of amino acid positions including: 153, 157, 159, 160, 162, 163, 164, 186, 189, and 194, wherein the substitutions and the positions are determined with reference to the sequence of SEQ ID NO:9. [This invention 1045] 1044. The polypeptide of the present invention, wherein the modified CH3 domain comprises Glu or Trp at position 153, Val, Trp, Leu, or Tyr at position 157, Leu, Pro, Phe, Thr, or His at position 159, Pro, Val, or Glu at position 160, Ala, Ser, Val, or Gly at position 162, Leu, His, Gln, Gly, Val, Ala, Asn, Asp, Thr, or Glu at position 163, Thr, Phe, Gln, Val, or Tyr at position 164, Leu, Ser, Glu, Ala, or Pro at position 186, Glu, Asp, Thr, or Asn at position 189, and Trp, Tyr, Phe, or His at position 194. [The present invention 1046] 1045. The polypeptide of the invention, wherein the modified CH3 domain comprises Glu or Trp at position 153, Trp, Leu, or Tyr at position 157, Thr or His at position 159, Val at position 160, Ala, Ser, or Val at position 162, Val, Asn, or Thr at position 163, Gln or Tyr at position 164, Pro at position 186, Thr or Asn at position 189, and Trp, Tyr, Phe, or His at position 194. [This invention 1047] The polypeptide of any one of claims 1044 to 1046, wherein the modified CH3 domain has the sequence of SEQ ID NO: 577 or 580. [This invention 1048] 1. A polypeptide comprising a modified CH3 domain that specifically binds to a transferrin receptor, wherein the modified CH3 domain comprises 5, 6, 7, or 8 substitutions at a set of amino acid positions including positions 118, 119, 120, 122, 210, 211, 212, and 213, and the substitutions and positions are determined based on amino acids 114 to 220 of SEQ ID NO:1. [This invention 1049] 1048. The polypeptide of the invention, wherein said modified CH3 domain comprises a Gly at position 210, a Phe at position 211, and / or an Asp at position 213. [The present invention 1050] 10. The modified CH3 domain comprising: Position 118 is Phe or Ile, position 119 is Asp, Glu, Gly, Ala, or Lys, position 120 is Tyr, Met, Leu, Ile, or Asp, position 122 is Thr or Ala, position 210 is Gly, position 211 is Phe, position 212 is His, Tyr, Ser, or Phe, and position 213 is Asp 1048. A polypeptide of the present invention, comprising at least one position selected from: [This invention 1051] 10. The modified CH3 domain comprising: Position 118 is Phe or Ile, position 119 is Asp, Glu, Gly, Ala, or Lys, position 120 is Tyr, Met, Leu, Ile, or Asp, position 122 is Thr or Ala, position 210 is Gly, position 211 is Phe, position 212 is His, Tyr, Ser, or Phe, and position 213 is Asp 1050. The polypeptide of the present invention, comprising two, three, four, five, six, seven, or eight positions selected from: [This invention 1052] The polypeptide of any of claims 1048 to 1051, wherein the modified CH3 domain has at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 114 to 220 of any one of SEQ ID NOs: 30 to 46. [This invention 1053] Any of the polypeptides of claims 1048 to 1051, wherein the modified CH3 domain has at least 85% identity to amino acids 114 to 220 of SEQ ID NO:1, with the proviso that the set of positions 118, 119, 120, 122, 210, 211, 212, and 213 are not included in the percent identity. [This invention 1054] 1048-1053. The polypeptide of any one of claims 1048-1053, wherein the modified CH3 domain comprises amino acids 118-122 and / or 210-213 of any one of SEQ ID NOs: 30-46. [This invention 1055] Any of the polypeptides of the present invention 1034 to 1054, wherein the modified CH3 domain further comprises (i) Trp at position 139, or (ii) Ser at position 139, Ala at position 141, and Val at position 180, and the amino acid positions are determined based on SEQ ID NO:1. [The present invention 1056] Any of the polypeptides of the present invention 1034 to 1055, wherein the modified CH3 domain further comprises (i) Leu at position 201 and Ser at position 207, or (ii) Ser or Ala at position 207, and the amino acid positions are determined based on SEQ ID NO:1. [This invention 1057] The polypeptide of any of claims 1001 to 1056, wherein the corresponding unmodified CH3 domain is a CH3 domain of human IgG1, IgG2, IgG3, or IgG4. [This invention 1058] The polypeptide of any one of claims 1001 to 1057, which is linked to a CH2 domain. [This invention 1059] The CH2 domain has the following set of modifications based on the amino acid sequence of SEQ ID NO:1: (a) Ala at positions 7 and 8, and (b) Tyr at position 25, Thr at position 27, and Glu at position 29 1058. A polypeptide of the present invention comprising one or both of: [The present invention 1060] 1059. The polypeptide of the present invention, wherein said set (a) further comprises Gly at position 102. [The present invention 1061] The polypeptide of any one of 1058 to 1060, wherein the CH2 domain is a CH2 domain of human IgG1, IgG2, IgG3, or IgG4. [The present invention 1062] The polypeptide of any one of 1058 to 1061 of the present invention, further linked to Fab. [The present invention 1063] The polypeptide of any of claims 1058 to 1062, wherein said polypeptide is a first polypeptide of a dimer such that the dimer is monovalent for binding to the transferrin receptor. [This invention 1064] The polypeptide of any of claims 1058 to 1062, wherein the polypeptide is a first polypeptide that binds to the transferrin receptor and forms a dimer with a second polypeptide comprising a modified CH3 domain. [This invention 1065] The polypeptide of claim 1064, wherein the modified CH3 domain of said second polypeptide is the same as the modified CH3 domain of said first polypeptide. [The present invention 1066] 1. A polypeptide comprising a modified CH2 domain that specifically binds to a transferrin receptor, wherein the modified CH2 domain comprises 5, 6, 7, 8, or 9 substitutions at a set of amino acid positions including positions 47, 49, 56, 58, 59, 60, 61, 62, and 63, and the substitutions and positions are determined based on amino acids 4 to 113 of SEQ ID NO:1. [This invention 1067] 1066. The polypeptide of the invention, wherein said modified CH2 domain comprises Glu at position 60 and / or Trp at position 61. [The present invention 1068] 10. The modified CH2 domain comprising: Position 47 is Glu, Gly, Gln, Ser, Ala, Asn, Tyr, or Trp; position 49 is Ile, Val, Asp, Glu, Thr, Ala, or Tyr; position 56 is Asp, Pro, Met, Leu, Ala, Asn, or Phe; position 58 is Arg, Ser, Ala, or Gly; position 59 is Tyr, Trp, Arg, or Val; position 60 is Glu; position 61 is Trp or Tyr; position 62 is Gln, Tyr, His, Ile, Phe, Val, or Asp; and position 63 is Leu, Trp, Arg, Asn, Tyr, or Val. 1066. A polypeptide of the present invention, comprising at least one position selected from: [The present invention 1069] 10. The modified CH2 domain comprising: Position 47 is Glu, Gly, Gln, Ser, Ala, Asn, Tyr, or Trp; position 49 is Ile, Val, Asp, Glu, Thr, Ala, or Tyr; position 56 is Asp, Pro, Met, Leu, Ala, Asn, or Phe; position 58 is Arg, Ser, Ala, or Gly; position 59 is Tyr, Trp, Arg, or Val; position 60 is Glu; position 61 is Trp or Tyr; position 62 is Gln, Tyr, His, Ile, Phe, Val, or Asp; and position 63 is Leu, Trp, Arg, Asn, Tyr, or Val. 1068. The polypeptide of the present invention, comprising at least two, three, four, five, six, seven, eight, or nine positions selected from: [The present invention 1070] 1068 or 1069, wherein the modified CH2 domain comprises Glu, Gly, Gln, Ser, Ala, Asn, or Tyr at position 47, Ile, Val, Asp, Glu, Thr, Ala, or Tyr at position 49, Asp, Pro, Met, Leu, Ala, or Asn at position 56, Arg, Ser, or Ala at position 58, Tyr, Trp, Arg, or Val at position 59, Glu at position 60, Trp at position 61, Gln, Tyr, His, Ile, Phe, or Val at position 62, and / or Leu, Trp, Arg, Asn, or Tyr at position 63. [This invention 1071] The polypeptide of any of claims 1068 to 1070, wherein the modified CH2 domain comprises Arg at position 58, Tyr or Trp at position 59, Glu at position 60, Trp at position 61, and / or Arg or Trp at position 63. [This invention 1072] The polypeptide of any of claims 1066 to 1071, wherein the modified CH2 domain has at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 4 to 113 of any one of SEQ ID NOs: 47 to 62. [This invention 1073] Any of polypeptides 1066 to 1071 of the present invention, wherein the modified CH2 domain has at least 85% identity with amino acids 4 to 113 of SEQ ID NO:1, with the proviso that the set of positions 47, 49, 56, 58, 59, 60, 61, 62, and 63 is not included in the percent identity. [This invention 1074] The polypeptide of any one of claims 1066 to 1073, wherein the modified CH2 domain comprises amino acids 47 to 49 and / or 56 to 63 of any one of SEQ ID NOs: 47 to 62. [This invention 1075] 1. A polypeptide comprising a modified CH2 domain that specifically binds to a transferrin receptor, wherein the modified CH2 domain comprises 5, 6, 7, 8, 9, or 10 substitutions at a set of amino acid positions including positions 39, 40, 41, 42, 43, 44, 68, 70, 71, and 72, and the substitutions and positions are determined based on amino acids 4 to 113 of SEQ ID NO:1. [This invention 1076] 1075. The polypeptide of the invention, wherein said modified CH2 domain comprises Pro at position 43, Glu at position 68, and / or Tyr at position 70. [This invention 1077] 10. The modified CH2 domain comprising: Position 39 is Pro, Phe, Ala, Met, or Asp; position 40 is Gln, Pro, Arg, Lys, Ala, Ile, Leu, Glu, Asp, or Tyr; position 41 is Thr, Ser, Gly, Met, Val, Phe, Trp, or Leu; position 42 is Pro, Val, Ala, Thr, or Asp; position 43 is Pro, Val, or Phe; position 44 is Trp, Gln, Thr, or Glu; position 68 is Glu, Val, Thr, Leu, or Trp; position 70 is Tyr, His, Val, or Asp; position 71 is Thr, His, Gln, Arg, Asn, or Val; and position 72 is Tyr, Asn, Asp, Ser, or Pro. 1075. A polypeptide of the present invention comprising at least one position selected from: [This invention 1078] 10. The modified CH2 domain comprising: Position 39 is Pro, Phe, Ala, Met, or Asp; position 40 is Gln, Pro, Arg, Lys, Ala, Ile, Leu, Glu, Asp, or Tyr; position 41 is Thr, Ser, Gly, Met, Val, Phe, Trp, or Leu; position 42 is Pro, Val, Ala, Thr, or Asp; position 43 is Pro, Val, or Phe; position 44 is Trp, Gln, Thr, or Glu; position 68 is Glu, Val, Thr, Leu, or Trp; position 70 is Tyr, His, Val, or Asp; position 71 is Thr, His, Gln, Arg, Asn, or Val; and position 72 is Tyr, Asn, Asp, Ser, or Pro. 1077. The polypeptide of the present invention, comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions selected from: [This invention 1079] The polypeptide of the present invention 1077 or 1078, wherein the modified CH2 domain comprises Pro, Phe, or Ala at position 39, Gln, Pro, Arg, Lys, Ala, or Ile at position 40, Thr, Ser, Gly, Met, Val, Phe, or Trp at position 41, Pro, Val, or Ala at position 42, Pro at position 43, Trp or Gln at position 44, Glu at position 68, Tyr at position 70, Thr, His, or Gln at position 71, and / or Tyr, Asn, Asp, or Ser at position 72. [The present invention 1080] The polypeptide of the invention 1077 or 1078, wherein the modified CH2 domain comprises Met at position 39, Leu or Glu at position 40, Trp at position 41, Pro at position 42, Val at position 43, Thr at position 44, Val or Thr at position 68, His at position 70, His, Arg, or Asn at position 71, and / or Pro at position 72. [This invention 1081] The polypeptide of the invention 1077 or 1078, wherein the modified CH2 domain comprises Asp at position 39, Asp at position 40, Leu at position 41, Thr at position 42, Phe at position 43, Gln at position 44, Val or Leu at position 68, Val at position 70, Thr at position 71, and / or Pro at position 72. [This invention 1082] The polypeptide of any of claims 1075 to 1081, wherein the modified CH2 domain has at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 4 to 113 of any one of SEQ ID NOs: 63 to 85. [This invention 1083] Any of the polypeptides of the present invention 1075 to 1081, wherein the modified CH2 domain has at least 85% identity with amino acids 4 to 113 of SEQ ID NO: 1, with the proviso that the set of positions 39, 40, 41, 42, 43, 44, 68, 70, 71, and 72 are not included in the percent identity. [This invention 1084] The polypeptide of any one of claims 1075 to 1083, wherein the modified CH2 domain comprises amino acids 39 to 44 and / or 68 to 72 of any one of SEQ ID NOs: 63 to 85. [This invention 1085] 1. A polypeptide comprising a modified CH2 domain that specifically binds to a transferrin receptor, wherein the modified CH2 domain comprises 5, 6, 7, 8, 9, or 10 substitutions at a set of amino acid positions including positions 41, 42, 43, 44, 45, 65, 66, 67, 69, and 73, and the substitutions and positions are determined based on amino acids 4 to 113 of SEQ ID NO:1. [The present invention 1086] 10. The modified CH2 domain comprising: Val or Asp at position 41, Pro, Met, or Asp at position 42, Pro or Trp at position 43, Arg, Trp, Glu, or Thr at position 44, Met, Tyr, or Trp at position 45, Leu or Trp at position 65, Thr, Val, Ile, or Lys at position 66, Ser, Lys, Ala, or Leu at position 67, His, Leu, or Pro at position 69, and Val or Trp at position 73. 1085. A polypeptide of the present invention, comprising at least one position selected from: [This invention 1087] 10. The modified CH2 domain comprising: Val or Asp at position 41, Pro, Met, or Asp at position 42, Pro or Trp at position 43, Arg, Trp, Glu, or Thr at position 44, Met, Tyr, or Trp at position 45, Leu or Trp at position 65, Thr, Val, Ile, or Lys at position 66, Ser, Lys, Ala, or Leu at position 67, His, Leu, or Pro at position 69, and Val or Trp at position 73 1086. A polypeptide of the invention comprising two, three, four, five, six, seven, eight, nine, or ten positions selected from: [This invention 1088] The polypeptide of the present invention 1086 or 1087, wherein the modified CH2 domain comprises Val at position 41, Pro at position 42, Pro at position 43, Arg or Trp at position 44, Met at position 45, Leu at position 65, Thr at position 66, Ser at position 67, His at position 69, and / or Val at position 73. [This invention 1089] The polypeptide of the present invention 1086 or 1087, wherein the modified CH2 domain comprises Asp at position 41, Met or Asp at position 42, Trp at position 43, Glu or Thr at position 44, Tyr or Trp at position 45, Trp at position 65, Val, Ile, or Lys at position 66, Lys, Ala, or Leu at position 67, Leu or Pro at position 69, and / or Trp at position 73. [The present invention 1090] The polypeptide of any of claims 1085 to 1089, wherein the modified CH2 domain has at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 4 to 113 of any one of SEQ ID NOs: 86 to 90. [This invention 1091] Any of the polypeptides of 1085 to 1089 of the present invention, wherein the modified CH2 domain has at least 85% identity with amino acids 4 to 113 of SEQ ID NO: 1, with the proviso that the set of positions 41, 42, 43, 44, 45, 65, 66, 67, 69, and 73 are not included in the percent identity. [This invention 1092] 1085-1091, the polypeptide of any one of claims 1085-1091, wherein the modified CH2 domain comprises amino acids 41-45 and / or 65-73 of any one of SEQ ID NOs: 86-90. [This invention 1093] 1. A polypeptide comprising a modified CH2 domain that specifically binds to a transferrin receptor, wherein the modified CH2 domain comprises 5, 6, 7, 8, or 9 substitutions at a set of amino acid positions including positions 45, 47, 49, 95, 97, 99, 102, 103, and 104, and the substitutions and positions are determined based on amino acids 4 to 113 of SEQ ID NO:1. [This invention 1094] The polypeptide of claim 1093, wherein said modified CH2 domain comprises Trp at position 103. [This invention 1095] 10. The modified CH2 domain comprising: Position 45: Trp, Val, Ile, or Ala; position 47: Trp or Gly; position 49: Tyr, Arg, or Glu; position 95: Ser, Arg, or Gln; position 97: Val, Ser, or Phe; position 99: Ile, Ser, or Trp; position 102: Trp, Thr, Ser, Arg, or Asp; position 103: Trp; and position 104: Ser, Lys, Arg, or Val. The polypeptide of the present invention 1093 or 1094, comprising at least one position selected from: [This invention 1096] 10. The modified CH2 domain comprising: Position 45: Trp, Val, Ile, or Ala; position 47: Trp or Gly; position 49: Tyr, Arg, or Glu; position 95: Ser, Arg, or Gln; position 97: Val, Ser, or Phe; position 99: Ile, Ser, or Trp; position 102: Trp, Thr, Ser, Arg, or Asp; position 103: Trp; and position 104: Ser, Lys, Arg, or Val. 1095. The polypeptide of the present invention, comprising two, three, four, five, six, seven, eight, or nine positions selected from: [This invention 1097] The polypeptide of the present invention 1093 or 1094, wherein the modified CH2 domain comprises Val or Ile at position 45, Gly at position 47, Arg at position 49, Arg at position 95, Ser at position 97, Ser at position 99, Thr, Ser, or Arg at position 102, Trp at position 103, and / or Lys or Arg at position 104. [This invention 1098] The polypeptide of any of claims 1093 to 1097, wherein the modified CH2 domain has at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 4 to 113 of any one of SEQ ID NOs: 91 to 95. [This invention 1099] Any of polypeptides 1093 to 1097 of the present invention, wherein the modified CH2 domain has at least 85% identity with amino acids 4 to 113 of SEQ ID NO: 1, with the proviso that the set of positions 45, 47, 49, 95, 97, 99, 102, 103, and 104 are not included in the percent identity. [The present invention 1100] 1093-1099. The polypeptide of any one of claims 1093-1099, wherein the modified CH2 domain comprises amino acids 45-49 and / or 95-104 of any one of SEQ ID NOs: 91-95. [The present invention 1101] The polypeptide of any of claims 1066 to 1100, wherein the corresponding unmodified CH2 domain is the CH2 domain of human IgG1, IgG2, IgG3, or IgG4. [The present invention 1102] The modified CH2 domain has the following set of modifications based on the amino acid sequence of SEQ ID NO:1: (a) Ala at positions 7 and 8, and (b) Tyr at position 25, Thr at position 27, and Glu at position 29 A polypeptide of any one of 1066 to 1101 of the present invention, comprising one or both of the following: [The present invention 1103] The polypeptide of the present invention 1102, wherein the set (a) further comprises Gly at position 102. [The present invention 1104] The polypeptide of any one of 1066 to 1103 of the present invention, which is linked to a CH3 domain. [This invention 1105] The polypeptide of the present invention 1104, wherein the CH3 domain comprises (i) Trp at position 139, or (ii) Ser at position 139, Ala at position 141, and Val at position 180, and the amino acid positions are determined based on SEQ ID NO:1. [The present invention 1106] The polypeptide of the present invention 1104 or 1105, wherein the CH3 domain further comprises (i) Leu at position 201 and Ser at position 207, or (ii) Ser or Ala at position 207, the amino acid positions being determined based on SEQ ID NO:1. [This invention 1107] The polypeptide of any one of 1104 to 1106 of the present invention, further linked to Fab. [This invention 1108] 1108. The polypeptide of any of claims 1104 to 1107, wherein said polypeptide is a first polypeptide of a dimer such that the dimer is monovalent for binding to the transferrin receptor. [This invention 1109] The polypeptide of any one of claims 1104 to 1107, wherein the polypeptide is a first polypeptide that binds to the transferrin receptor and forms a dimer with a second polypeptide comprising a modified CH2 domain. [The present invention 1110] 1109. The polypeptide of claim 1109, wherein the modified CH2 domain of said second polypeptide is the same as the modified CH2 domain of said first polypeptide. [The present invention 1111] A polypeptide that specifically binds to a transferrin receptor, Amino acids 157 to 194 of any one of SEQ ID NOs: 4 to 29, 236 to 299, and 422 to 435; Amino acids 118 to 213 of any one of SEQ ID NOs: 30 to 46; Amino acids 47 to 63 of any one of SEQ ID NOs: 47 to 62; Amino acids 39 to 72 of any one of SEQ ID NOs: 63 to 85; Amino acids 41-73 of any one of SEQ ID NOs: 86-90, or Amino acids 45 to 104 of any one of SEQ ID NOs: 91 to 95 wherein the amino acid positions are determined with reference to SEQ ID NO:1. [The present invention 1112] A polypeptide that specifically binds to a transferrin receptor, a knob mutation and a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs: 349, 361, 373, 385, 397, 409, 436, 448, 460, and 472; the knob mutation is T139W when numbered with reference to SEQ ID NO:1; The polypeptide. [The present invention 1113] A polypeptide that specifically binds to a transferrin receptor, a knob mutation, a mutation that modulates effector function, and a sequence that has at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs: 350, 362, 374, 386, 398, 410, 437, 449, 461, and 473; When numbered based on SEQ ID NO: 1, the knob mutation is T139W and the effector function-regulating mutations are L7A and L8A. The polypeptide. [This invention 1114] A polypeptide that specifically binds to a transferrin receptor, a knob mutation, a mutation that modulates effector function, and a sequence that has at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs: 351, 363, 375, 387, 399, 411, 438, 450, 462, and 474; When numbered based on SEQ ID NO: 1, the knob mutation is T139W and the effector function-regulating mutations are L7A, L8A, and P102G. The polypeptide. [This invention 1115] A polypeptide that specifically binds to a transferrin receptor, a knob mutation, a mutation that increases serum stability, and a sequence that has at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs: 352, 364, 376, 388, 400, 412, 439, 451, 463, and 475; When numbered based on SEQ ID NO: 1, the knob mutation is T139W and the serum stability-enhancing mutations are M25Y, S27T, and T29E. The polypeptide. [The present invention 1116] A polypeptide that specifically binds to a transferrin receptor, a knob mutation, a mutation that increases serum stability, and a sequence that has at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs: 485, 492, 499, 506, 513, 520, 527, 534, 541, and 548; When numbered based on SEQ ID NO: 1, the knob mutation is T139W and the serum stability-enhancing mutations are M201L and N207S. The polypeptide. [This invention 1117] A polypeptide that specifically binds to a transferrin receptor, a knob mutation, a mutation that modulates effector function, a mutation that increases serum stability, and a sequence that has at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs: 353, 365, 377, 389, 401, 413, 440, 452, 464, and 476; When numbered based on SEQ ID NO: 1, the knob mutation is T139W, the effector function-modulating mutations are L7A and L8A, and the serum stability-enhancing mutations are M25Y, S27T, and T29E. The polypeptide. [This invention 1118] A polypeptide that specifically binds to a transferrin receptor, a knob mutation, a mutation that modulates effector function, a mutation that increases serum stability, and a sequence that has at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs: 486, 493, 500, 507, 514, 521, 528, 535, 542, and 549; When numbered based on SEQ ID NO: 1, the knob mutation is T139W, the effector function-modulating mutations are L7A and L8A, and the serum stability-enhancing mutations are M201L and N207S. The polypeptide. [This invention 1119] A polypeptide that specifically binds to a transferrin receptor, a knob mutation, a mutation that modulates effector function, a mutation that increases serum stability, and a sequence that has at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs: 354, 366, 378, 390, 402, 414, 441, 453, 465, and 477; When numbered based on SEQ ID NO: 1, the knob mutation is T139W, the effector function-modulating mutations are L7A, L8A, and P102G, and the serum stability-enhancing mutations are M25Y, S27T, and T29E. The polypeptide. [The present invention 1120] A polypeptide that specifically binds to a transferrin receptor, a knob mutation, a mutation that modulates effector function, a mutation that increases serum stability, and a sequence that has at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs: 487, 494, 501, 508, 515, 522, 529, 536, 543, and 550; When numbered based on SEQ ID NO: 1, the knob mutation is T139W, the effector function-modulating mutations are L7A, L8A, and P102G, and the serum stability-enhancing mutations are M201L and N207S. The polypeptide. [This invention 1121] A polypeptide that specifically binds to a transferrin receptor, a hole mutation and a sequence having at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs: 355, 367, 379, 391, 403, 415, 442, 454, 466, and 478; When numbered based on SEQ ID NO: 1, the hole mutations are T139S, L141A, and Y180V; The polypeptide. [This invention 1122] A polypeptide that specifically binds to a transferrin receptor, a hole mutation, a mutation that modulates effector function, and a sequence that has at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs: 356, 368, 380, 392, 404, 416, 443, 455, 467, and 479; When numbered based on SEQ ID NO: 1, the hole mutations are T139S, L141A, and Y180V, and the effector function-regulating mutations are L7A and L8A. The polypeptide. [This invention 1123] A polypeptide that specifically binds to a transferrin receptor, a hole mutation, a mutation that modulates effector function, and a sequence that has at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs: 357, 369, 381, 393, 405, 417, 444, 456, 468, and 480; When numbered based on SEQ ID NO: 1, the hole mutations are T139S, L141A, and Y180V, and the effector function-regulating mutations are L7A, L8A, and P102G. The polypeptide. [This invention 1124] A polypeptide that specifically binds to a transferrin receptor, a whole mutation, a mutation that increases serum stability, and a sequence that has at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs: 358, 370, 382, ​​394, 406, 418, 445, 457, 469, and 481; When numbered based on SEQ ID NO: 1, the hole mutations are T139S, L141A, and Y180V, and the serum stability-enhancing mutations are M25Y, S27T, and T29E. The polypeptide. [Invention 1125] A polypeptide that specifically binds to a transferrin receptor, a whole mutation, a mutation that increases serum stability, and a sequence that has at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs: 488, 495, 502, 509, 516, 523, 530, 537, 544, and 551; When numbered based on SEQ ID NO: 1, the hole mutations are T139S, L141A, and Y180V, and the serum stability-enhancing mutations are M201L and N207S. The polypeptide. [The present invention 1126] A polypeptide that specifically binds to a transferrin receptor, a whole mutation, a mutation that modulates effector function, a mutation that increases serum stability, and a sequence that has at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs: 359, 371, 383, 395, 407, 419, 446, 458, 470, and 482; When numbered based on SEQ ID NO: 1, the hole mutations are T139S, L141A, and Y180V, the effector function-regulating mutations are L7A and L8A, and the serum stability-enhancing mutations are M25Y, S27T, and T29E. The polypeptide. [This invention 1127] A polypeptide that specifically binds to a transferrin receptor, a whole mutation, a mutation that modulates effector function, a mutation that increases serum stability, and a sequence that has at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs: 489, 496, 503, 510, 517, 524, 531, 538, 545, and 552; When numbered based on SEQ ID NO: 1, the hole mutations are T139S, L141A, and Y180V, the effector function-regulating mutations are L7A and L8A, and the serum stability-enhancing mutations are M201L and N207S. The polypeptide. [This invention 1128] A polypeptide that specifically binds to a transferrin receptor, a whole mutation, a mutation that modulates effector function, a mutation that increases serum stability, and a sequence that has at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs: 360, 372, 384, 396, 408, 420, 447, 459, 471, and 483; When numbered based on SEQ ID NO: 1, the hole mutations are T139S, L141A, and Y180V, the effector function-regulating mutations are L7A, L8A, and P102G, and the serum stability-enhancing mutations are M25Y, S27T, and T29E. The polypeptide. [This invention 1129] A polypeptide that specifically binds to a transferrin receptor, a whole mutation, a mutation that modulates effector function, a mutation that increases serum stability, and a sequence that has at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs: 490, 497, 504, 511, 518, 525, 532, 539, 546, and 553; When numbered based on SEQ ID NO: 1, the hole mutations are T139S, L141A, and Y180V, the effector function-regulating mutations are L7A, L8A, and P102G, and the serum stability-enhancing mutations are M201L and N207S. The polypeptide. [The present invention 1130] A polypeptide that specifically binds to a transferrin receptor, comprising any one of SEQ ID NOs: 116-233, 303-345, and 581-608. [This invention 1131] A polypeptide that specifically binds to a transferrin receptor, comprising a first sequence of any one of SEQ ID NOs:116-130 and a second sequence independently selected from the group consisting of SEQ ID NOs:131-139. [This invention 1132] A polypeptide of the present invention 1131 comprising a first sequence of any one of SEQ ID NOs: 121, 116, 122, 123, or 126-130, and a second sequence independently selected from the group consisting of SEQ ID NOs: 136, 137, and 139. [This invention 1133] A polypeptide of the present invention 1131 comprising a first sequence of any one of SEQ ID NO: 120 or 124 to 126 and a second sequence independently selected from the group consisting of SEQ ID NO: 135, 138, and 139. [This invention 1134] a) SEQ ID NO: 116 and SEQ ID NO: 131; b) SEQ ID NO: 116 and SEQ ID NO: 136; c) SEQ ID NO: 117 and SEQ ID NO: 132; d) SEQ ID NO: 118 and SEQ ID NO: 133; e) SEQ ID NO: 119 and SEQ ID NO: 134; f) SEQ ID NO: 120 and SEQ ID NO: 135; g) SEQ ID NO: 121 and SEQ ID NO: 136; h) SEQ ID NO: 122 and SEQ ID NO: 137; i) SEQ ID NO: 123 and SEQ ID NO: 136; j) SEQ ID NO: 124 and SEQ ID NO: 138; k) SEQ ID NO: 125 and SEQ ID NO: 135; l) SEQ ID NO: 126 and SEQ ID NO: 139; m) SEQ ID NO: 127 and SEQ ID NO: 136; n) SEQ ID NO: 128 and SEQ ID NO: 136; o) SEQ ID NO: 129 and SEQ ID NO: 136, or p) SEQ ID NO: 130 and SEQ ID NO: 136 1131. The polypeptide of the present invention comprising: [This invention 1135] A polypeptide that specifically binds to a transferrin receptor, comprising a first sequence of any one of SEQ ID NOs: 303 to 339 and a second sequence independently selected from the group consisting of SEQ ID NOs: 136, 138, and 340 to 345. [This invention 1136] a) SEQ ID NO: 303 and SEQ ID NO: 340; b) SEQ ID NO:304 and SEQ ID NO:340; c) SEQ ID NO: 305 and SEQ ID NO: 340; d) SEQ ID NO: 306 and SEQ ID NO: 341; e) SEQ ID NO: 307 and SEQ ID NO: 340; f) SEQ ID NO: 308 and SEQ ID NO: 340; g) SEQ ID NO: 309 and SEQ ID NO: 340; h) SEQ ID NO: 310 and SEQ ID NO: 340; i) SEQ ID NO: 311 and SEQ ID NO: 340; j) SEQ ID NO: 312 and SEQ ID NO: 341; k) SEQ ID NO: 313 and SEQ ID NO: 340; l) SEQ ID NO: 314 and SEQ ID NO: 340; m) SEQ ID NO: 315 and SEQ ID NO: 340; n) SEQ ID NO: 316 and SEQ ID NO: 340; o) SEQ ID NO: 317 and SEQ ID NO: 340; p) SEQ ID NO: 318 and SEQ ID NO: 341; q) SEQ ID NO: 319 and SEQ ID NO: 340; r) SEQ ID NO: 320 and SEQ ID NO: 340; s) SEQ ID NO: 321 and SEQ ID NO: 340; t) SEQ ID NO: 322 and SEQ ID NO: 340; u) SEQ ID NO: 323 and SEQ ID NO: 340; v) SEQ ID NO: 324 and SEQ ID NO: 341; w) SEQ ID NO: 325 and SEQ ID NO: 340; x) SEQ ID NO: 326 and SEQ ID NO: 340; y) SEQ ID NO: 327 and SEQ ID NO: 340; z) SEQ ID NO: 328 and SEQ ID NO: 340; aa) SEQ ID NO: 329 and SEQ ID NO: 340; ab) SEQ ID NO: 330 and SEQ ID NO: 341; ac) SEQ ID NO: 331 and SEQ ID NO: 340; ad) SEQ ID NO: 332 and SEQ ID NO: 340; ae) SEQ ID NO: 306 and SEQ ID NO: 340; af) SEQ ID NO: 312 and SEQ ID NO: 340; ag) SEQ ID NO: 324 and SEQ ID NO: 138; ah) SEQ ID NO: 318 and SEQ ID NO: 340; ai) SEQ ID NO: 324 and SEQ ID NO: 340; aj) SEQ ID NO: 330 and SEQ ID NO: 340; a) SEQ ID NO: 318 and SEQ ID NO: 138; al) SEQ ID NO: 333 and SEQ ID NO: 136; am) SEQ ID NO: 334 and SEQ ID NO: 136, an) SEQ ID NO:312 and SEQ ID NO:138, ao) SEQ ID NO: 333 and SEQ ID NO: 342, ap) SEQ ID NO: 335 and SEQ ID NO: 342, aq) SEQ ID NO: 336 and SEQ ID NO: 342, ar) SEQ ID NO: 334 and SEQ ID NO: 342, as) SEQ ID NO: 330 and SEQ ID NO: 138, at) SEQ ID NO: 330 and SEQ ID NO: 343, au) SEQ ID NO: 330 and SEQ ID NO: 345, av) SEQ ID NO: 337 and SEQ ID NO: 136, aw) SEQ ID NO: 338 and SEQ ID NO: 136, ax) SEQ ID NO: 339 and SEQ ID NO: 136; ay) SEQ ID NO: 330 and SEQ ID NO: 344, a-z) SEQ ID NO: 312 and SEQ ID NO: 343, or ba) SEQ ID NO: 312 and SEQ ID NO: 345 1135. A polypeptide of the present invention comprising: [This invention 1137] A polypeptide that specifically binds to a transferrin receptor, comprising a first sequence of any one of SEQ ID NOs:581, 583, 585, 587, 589, 591, and 593, and a second sequence independently selected from the group consisting of SEQ ID NOs:582, 884, 586, 588, 590, 592, and 594. [This invention 1138] a) SEQ ID NO: 581 and SEQ ID NO: 582; b) SEQ ID NO:583 and SEQ ID NO:584; c) SEQ ID NO: 585 and SEQ ID NO: 586; d) SEQ ID NO: 587 and SEQ ID NO: 588; e) SEQ ID NO: 589 and SEQ ID NO: 590; f) SEQ ID NO: 591 and SEQ ID NO: 592, or g) SEQ ID NO: 593 and SEQ ID NO: 594 The polypeptide of the present invention 1137, comprising: [This invention 1139] A polypeptide that specifically binds to a transferrin receptor, comprising a first sequence of any one of SEQ ID NOs:554, 557, and 560, and a second sequence independently selected from the group consisting of SEQ ID NOs:555, 558, and 561. [The present invention 1140] a) SEQ ID NO: 554 and SEQ ID NO: 555; b) SEQ ID NO: 557 and SEQ ID NO: 558, or c) SEQ ID NO: 560 and SEQ ID NO: 561 1139. A polypeptide of the present invention comprising: [This invention 1141] A polypeptide that specifically binds to a transferrin receptor, comprising a first sequence of any one of SEQ ID NOs:554, 557, and 560, and a second sequence independently selected from the group consisting of SEQ ID NOs:340 to 345, 582, 584, 586, 588, 590, 592, and 594. [This invention 1142] A polypeptide that specifically binds to a transferrin receptor, comprising a first sequence selected from any one of SEQ ID NOs: 303 to 339, 581, 583, 585, 587, 589, 591, and 593, and a second sequence independently selected from the group consisting of SEQ ID NOs: 555, 558, and 561. [This invention 1143] A polypeptide that specifically binds to a transferrin receptor, comprising a first sequence of any one of SEQ ID NOs:609-614 and a second sequence independently selected from the group consisting of SEQ ID NOs:615-620. [This invention 1144] A polypeptide that specifically binds to a transferrin receptor, comprising a first sequence of any one of SEQ ID NOs: 303, 312, 315 to 318, and 328, and a second sequence independently selected from the group consisting of SEQ ID NOs: 135, 340, and 341. [Invention 1145] a) SEQ ID NO: 303 and SEQ ID NO: 340; b) SEQ ID NO: 316 and SEQ ID NO: 340; c) SEQ ID NO: 317 and SEQ ID NO: 340; d) SEQ ID NO: 318 and SEQ ID NO: 340; e) SEQ ID NO: 328 and SEQ ID NO: 341; f) SEQ ID NO: 318 and SEQ ID NO: 340; g) SEQ ID NO: 312 and SEQ ID NO: 340; h) SEQ ID NO: 303 and SEQ ID NO: 341; i) SEQ ID NO:316 and SEQ ID NO:135, or j) SEQ ID NO: 315 and SEQ ID NO: 341 The polypeptide of the present invention 1144, comprising: [Invention 1146] A polypeptide that specifically binds to a transferrin receptor, comprising a first sequence of any one of SEQ ID NOs:595, 597, 599, 601, 603, 605, and 607, and a second sequence independently selected from the group consisting of SEQ ID NOs:596, 598, 600, 602, 604, 606, and 608. [This invention 1147] a) SEQ ID NO:595 and SEQ ID NO:596; b) SEQ ID NO:597 and SEQ ID NO:598; c) SEQ ID NO:599 and SEQ ID NO:600; d) SEQ ID NO:601 and SEQ ID NO:602; e) SEQ ID NO:603 and SEQ ID NO:604; f) SEQ ID NO:605 and SEQ ID NO:606, or g) SEQ ID NO: 607 and SEQ ID NO: 608 1146. A polypeptide of the present invention comprising: [Invention 1148] A polypeptide that specifically binds to a transferrin receptor, comprising a first sequence of either SEQ ID NO:575 or 578 and a second sequence independently selected from the group consisting of SEQ ID NO:576 and 579. [This invention 1149] a) SEQ ID NO: 575 and SEQ ID NO: 576, or b) SEQ ID NO: 578 and SEQ ID NO: 579 1148. The polypeptide of the present invention comprising: [This invention 1150] A polypeptide that specifically binds to a transferrin receptor, comprising a first sequence of either SEQ ID NO:575 or 578 and a second sequence independently selected from the group consisting of SEQ ID NO:136, 138, and 340 to 345. [This invention 1151] A polypeptide that specifically binds to a transferrin receptor, comprising a first sequence selected from any one of SEQ ID NOs: 303 to 339 and a second sequence independently selected from the group consisting of SEQ ID NOs: 576 and 579. [This invention 1152] A polypeptide that specifically binds to a transferrin receptor, comprising a first sequence of any one of SEQ ID NOs:140 to 153 and a second sequence independently selected from the group consisting of SEQ ID NOs:154 to 157. [This invention 1153] a) SEQ ID NO: 140 and SEQ ID NO: 154; b) SEQ ID NO: 141 and SEQ ID NO: 154; c) SEQ ID NO: 142 and SEQ ID NO: 154; d) SEQ ID NO: 143 and SEQ ID NO: 154; e) SEQ ID NO: 144 and SEQ ID NO: 154; f) SEQ ID NO: 145 and SEQ ID NO: 154; g) SEQ ID NO: 146 and SEQ ID NO: 154; h) SEQ ID NO: 147 and SEQ ID NO: 154; i) SEQ ID NO: 148 and SEQ ID NO: 155; j) SEQ ID NO: 149 and SEQ ID NO: 154; k) SEQ ID NO: 140 and SEQ ID NO: 156; l) SEQ ID NO: 150 and SEQ ID NO: 156; m) SEQ ID NO: 151 and SEQ ID NO: 157; n) SEQ ID NO: 152 and SEQ ID NO: 155, or o) SEQ ID NO: 153 and SEQ ID NO: 154 1152. A polypeptide of the present invention comprising: [This invention 1154] A polypeptide that specifically binds to a transferrin receptor, comprising a first sequence of any one of SEQ ID NOs:158-171 and a second sequence independently selected from the group consisting of SEQ ID NOs:172-186. [Invention 1155] a) SEQ ID NO: 158 and SEQ ID NO: 172; b) SEQ ID NO: 158 and SEQ ID NO: 179; c) SEQ ID NO: 159 and SEQ ID NO: 173; d) SEQ ID NO: 159 and SEQ ID NO: 181; e) SEQ ID NO: 160 and SEQ ID NO: 174; f) SEQ ID NO: 161 and SEQ ID NO: 175; g) SEQ ID NO: 162 and SEQ ID NO: 176; h) SEQ ID NO: 163 and SEQ ID NO: 177; i) SEQ ID NO: 164 and SEQ ID NO: 178; j) SEQ ID NO: 165 and SEQ ID NO: 180; k) SEQ ID NO: 166 and SEQ ID NO: 182; l) SEQ ID NO: 167 and SEQ ID NO: 183; m) SEQ ID NO: 168 and SEQ ID NO: 184; n) SEQ ID NO: 169 and SEQ ID NO: 185; o) SEQ ID NO: 170 and SEQ ID NO: 174, or p) SEQ ID NO: 171 and SEQ ID NO: 186 1154. The polypeptide of the present invention comprising: [Invention 1156] A polypeptide that specifically binds to a transferrin receptor, comprising a first sequence of any one of SEQ ID NOs:187-204 and a second sequence independently selected from the group consisting of SEQ ID NOs:205-215. [This invention 1157] a) SEQ ID NO: 187 and SEQ ID NO: 205; b) SEQ ID NO: 187 and SEQ ID NO: 206; c) SEQ ID NO: 188 and SEQ ID NO: 206; d) SEQ ID NO: 189 and SEQ ID NO: 207; e) SEQ ID NO: 190 and SEQ ID NO: 206; f) SEQ ID NO: 191 and SEQ ID NO: 205; g) SEQ ID NO: 192 and SEQ ID NO: 206; h) SEQ ID NO: 193 and SEQ ID NO: 208; i) SEQ ID NO: 194 and SEQ ID NO: 206; j) SEQ ID NO: 195 and SEQ ID NO: 209; k) SEQ ID NO: 196 and SEQ ID NO: 206; l) SEQ ID NO: 197 and SEQ ID NO: 205; m) SEQ ID NO: 198 and SEQ ID NO: 206; n) SEQ ID NO: 199 and SEQ ID NO: 208; o) SEQ ID NO: 200 and SEQ ID NO: 206; p) SEQ ID NO: 201 and SEQ ID NO: 210; q) SEQ ID NO: 201 and SEQ ID NO: 211; r) SEQ ID NO: 201 and SEQ ID NO: 212; s) SEQ ID NO: 202 and SEQ ID NO: 212; t) SEQ ID NO: 203 and SEQ ID NO: 213; u) SEQ ID NO: 203 and SEQ ID NO: 214, or v) SEQ ID NO: 204 and SEQ ID NO: 215 1156. A polypeptide of the present invention comprising: [This invention 1158] A polypeptide that specifically binds to a transferrin receptor, comprising a first sequence of any one of SEQ ID NOs:216-220 and a second sequence independently selected from the group consisting of SEQ ID NOs:221-224. [This invention 1159] a) SEQ ID NO: 216 and SEQ ID NO: 221; b) SEQ ID NO: 217 and SEQ ID NO: 221; c) SEQ ID NO: 218 and SEQ ID NO: 222; d) SEQ ID NO: 219 and SEQ ID NO: 223, or e) SEQ ID NO: 220 and SEQ ID NO: 224 1158. A polypeptide of the present invention comprising: [The present invention 1160] A polypeptide that specifically binds to a transferrin receptor, comprising a first sequence of any one of SEQ ID NOs:225-228 and a second sequence independently selected from the group consisting of SEQ ID NOs:229-233. [This invention 1161] a) SEQ ID NO: 225 and SEQ ID NO: 229; b) SEQ ID NO: 226 and SEQ ID NO: 230; c) SEQ ID NO: 226 and SEQ ID NO: 231; d) SEQ ID NO: 227 and SEQ ID NO: 232, or e) SEQ ID NO: 228 and SEQ ID NO: 233 1160. A polypeptide of the present invention comprising: [This invention 1162] A polynucleotide comprising a nucleic acid sequence encoding any one of the polypeptides 1001 to 1161 of the present invention. [This invention 1163] A vector comprising the polynucleotide of the present invention. [Invention 1164] A host cell comprising a polynucleotide of the present invention. [This invention 1165] A method for producing a polypeptide comprising a modified CH3 domain or a modified CH2 domain, the method comprising culturing a host cell under conditions in which a polypeptide encoded by a polynucleotide of the present invention 1162 is expressed. [Invention 1166] A pharmaceutical composition comprising any one of the polypeptides of the present inventions 1001 to 1161 and a pharmaceutically acceptable carrier. [This invention 1167] A method for transcytosing a composition across an endothelium, the method comprising contacting the endothelium with a composition comprising any one of the polypeptides of the present inventions 1001 to 1161. [Invention 1168] 1167. The method of claim 1167, wherein said endothelium is the blood-brain barrier (BBB). [This invention 1169] 1. A method for engineering a CH3 domain to specifically bind to a transferrin receptor, comprising: (a) a polynucleotide encoding the CH3 domain, (i) 157, 159, 160, 161, 162, 163, 186, 189, and 194, or (ii) 118, 119, 120, 122, 210, 211, 212, and 213 and modifying the polypeptide to have at least five amino acid substitutions at a set of amino acid positions comprising: (b) expressing a polypeptide comprising the modified CH3 domain; (c) determining whether the modified CH3 domain binds to the transferrin receptor; and The method comprising: [This invention 1170] 1169. The method of claim 1169, wherein the steps of expressing said polypeptide comprising said modified CH3 domain and determining whether said modified CH3 domain binds to said transferrin receptor are carried out using a display system. [This invention 1171] 1170. The method of claim 1170, wherein said display system is a cell surface display system, a viral display system, an mRNA display system, a polysome display system, or a ribosome display system. [This invention 1172] 1169. The method of claim 1169, wherein said polypeptide comprising said modified CH3 domain is expressed as a soluble protein. [This invention 1173] 1. A method for engineering a CH2 domain to specifically bind to a transferrin receptor, comprising: (a) a polynucleotide encoding the CH2 domain, (i) 47, 49, 56, 58, 59, 60, 61, 62, and 63; (ii) 39, 40, 41, 42, 43, 44, 68, 70, 71, and 72; (iii) 41, 42, 43, 44, 45, 65, 66, 67, 69, and 73, or (iv) 45, 47, 49, 95, 97, 99, 102, 103, and 104 and modifying the polypeptide to have at least five amino acid substitutions at a set of amino acid positions comprising: (b) expressing a polypeptide comprising the modified CH2 domain; (c) determining whether the modified CH2 domain binds to the transferrin receptor; and The method comprising: [This invention 1174] 1173. The method of claim 1173, wherein the steps of expressing said polypeptide comprising said modified CH2 domain and determining whether said modified CH2 domain binds to said transferrin receptor are carried out using a display system. [This invention 1175] 1174. The method of claim 1174, wherein said display system is a cell surface display system, a viral display system, an mRNA display system, a polysome display system, or a ribosome display system. [Invention 1176] The method of claim 1173, wherein said polypeptide comprising said modified CH2 domain is expressed as a soluble protein. [This invention 1177] 1. A method for engineering a CH3 domain to specifically bind to a transferrin receptor, comprising: (a) modifying the polynucleotide encoding the CH3 domain so that it has at least five amino acid substitutions selected from the group consisting of 1001 to 1023, 1027 to 1029, 1032 to 1033, 1048 to 1051, and 1055 to 1056 of the present invention; (b) expressing and recovering a polypeptide comprising the modified CH3 domain; The method comprising: [This invention 1178] 1. A method for engineering a CH2 domain to specifically bind to a transferrin receptor, comprising: (a) modifying a polynucleotide encoding the CH2 domain so that it has at least five amino acid substitutions selected from the group consisting of 1066 to 1071, 1075 to 1081, 1085 to 1089, and 1093 to 1097 of the present invention; (b) expressing and recovering a polypeptide comprising the modified CH2 domain; The method comprising: [This invention 1179] 1. A method for enhancing binding of an engineered Fc polypeptide comprising a non-native binding site to a target, comprising: (a) introducing one or more substitutions at one or more positions within 10 Å of said non-native binding site; (b) testing the modified Fc polypeptide for binding to the target; The method comprising: [This invention 1180] The non-natural binding site is located at the following position: 157th, 159th, 160th, 161st, 162nd, 163rd, 186th, 189th, and 194th 1179. The method of claim 1179, comprising a substitution of one or more of: [This invention 1181] The one or more substitutions at one or more positions within 10 Å of the non-native binding site are The method of claim 1179 or 1180, wherein the nucleotide sequence is selected from the group consisting of K21, R28, Q115, R117, E118, Q120, T132, K133, N134, Q135, S137, K143, E153, E155, S156, G158, Y164, K165, T166, D172, S173, D174, S176, K182, L183, T184, V185, K187, S188, Q191, Q192, G193, V195, F196, S197, S199, Q211, S213, S215, L216, S217, P218, G219, and K220, based on NO:1. [This invention 1182] 1182. The method of any one of claims 1179 to 1181, wherein said target is a transferrin receptor. [Brief explanation of the drawings]

[0103] [Figure 1]The results of phage ELISA for four CH2A2 clones are shown. CH2A2 Fc variants were expressed on the surface of phage and tested for binding to plate-coated anti-c-Myc antibody 9E10 (expression control), negative control, human transferrin receptor (TfR), and cynomolgus monkey (cyno) TfR. The x-axis shows the OD268 of the phage solution, which is a measure of phage concentration. (A) ELISA results for clone CH2A2.5 are shown. (B) ELISA results for clone CH2A2.1 are shown. (C) ELISA results for clone CH2A2.4 are shown. (D) ELISA results for clone CH2A2.16 are shown. [Figure 2] Figure 1 shows the results of a phage ELISA of the CH2A2 clone binding to human TfR. Phage were added to ELISA plates coated with TfR at approximately the binding EC50, and soluble holo-Tf or soluble TfR was added at different concentrations. The data show that the CH2A2 clone competed with soluble TfR, but not with holo-Tf, for binding to the TfR coated on the plate. (A) Results of the experiment in which soluble holo-Tf was added are shown. (B) Results of the experiment in which soluble TfR was added are shown. [Figure 3A]Figure 1 shows the binding of CH2C clones to TfR in the presence or absence of holo-Tf. (A) Phage ELISA results in which TfR was coated onto an ELISA plate and phage-displayed clone CH2C.23 was added in the presence or absence of a large excess of holo-Tf (5 μM). (B) Binding of CH2C clones in Fc-Fab fusion format to ELISA plates coated with human or cynomolgus TfR. (C) Phage ELISA results in which human TfR, cynomolgus TfR, holo-Tf, anti-Myc, or streptavidin was coated onto an ELISA plate and phage-displayed clones CH2C.17 and CH2C.22 were added at different dilutions in the presence or absence of holo-Tf. These data indicate that these clones did not compete with holo-Tf for binding to TfR. (D) Octet® (i.e., Biolayer Interferometry) kinetic trace of the binding of clone CH2C.7 to TfR-biotin coated on an anti-streptavidin sensor in the presence of 5 μM holo-Tf, with background subtracted for the binding of holo-Tf alone, showing no competition for binding with Tf. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 4]Binding of CH3B clones to TfR in the presence or absence of holo-Tf is shown. (A) Results of a phage ELISA in which human TfR, cynomolgus monkey TfR, holo-Tf, anti-Myc, or streptavidin were coated onto ELISA plates, and phage-displayed clones CH3B.11 and CH3B.12 were added at different dilutions in the presence or absence of holo-Tf. These data indicate that these clones did not compete with holo-Tf for binding to TfR. (B) Binding of CH3B clones to ELISA plates coated with human or cynomolgus monkey TfR is shown. The Fc region containing the CH3B clone sequence was fused to a Fab fragment and assayed in a dimer format. [Figure 5] NNK patch library for maturation of CH3B clones. Ribbons show the backbone of the CH3 domain, the dark surface represents the original CH3B register, and the light surface patches represent the expanded repertoire. [Figure 6] Figure 1 shows FACS plots of selection of CH3C clones in yeast, showing enrichment of the binding population after three sorting rounds. In sorting rounds 1 and 2, biotinylated TfR was preloaded with streptavidin-Alexa Fluor® 647 before incubation with yeast. In sorting round 3, biotinylated TfR was first incubated with yeast, and streptavidin-Alexa Fluor® 647 was added for secondary detection. In all sorting rounds, expression was monitored using a chicken anti-c-Myc antibody (obtained from Thermo Fisher) against the C-terminal Myc tag on the yeast display construct. [Figure 7]Binding of CH3C clones to TfR in the presence or absence of holo-Tf is shown. Clones were assayed in Fc-Fab fusion format. Ab204, a standard antibody with a variable region that binds to TfR, was used as a positive control for this assay. (A) Binding of CH3C mutants to human TfR coated on an ELISA plate is shown. (B) Binding of CH3C mutants to human TfR coated on an ELISA plate in the presence of 5 μM holo-Tf is shown. (C) Binding of CH3C mutants to cynomolgus monkey TfR coated on an ELISA plate is shown. [Figure 8] Figure 1 shows the binding of CH3C clones to 293F cells, which endogenously express the human TfR. Cells were distributed into 96-well V-bottom plates, and different concentrations of CH3C clones formatted as Fc-Fab fusion binding proteins were added. After 1 hour of incubation at 4°C, each plate was centrifuged, washed, and then incubated with goat anti-human IgG-Alexa Fluor® 647 secondary antibody for 30 minutes at 4°C. After further washing of the cells, each plate was read on a FACSCanto™ II flow cytometer, and the median fluorescence in the APC (647 nm) channel was determined using FlowJo® software. [Figure 9] Figure 1 shows the internalization of CH3C.3 into HEK293 cells that endogenously express the human TfR. After adding 1 μM CH3C.3 or a control for 30 minutes at 37°C and 8% CO2, the cells were washed, permeabilized, and stained with anti-human IgG-Alexa Fluor® 488 secondary antibody. After further washing, the cells were imaged by fluorescence microscopy, and the number of puncta was quantified. (A) Microscopy data is shown. (B) A graph of the number of puncta per well is shown. [Figure 10]The selection scheme for the CH3C soft library is shown. The initial library was sorted by MACS against either human (H) or cynomolgus (C) TfR. The resulting yeast pool was then split, and each was sorted against either human or cynomolgus TfR, similar to the first FAS sorting round. Each resulting pool was split again for another round of sorting. Finally, the HHH and CCC pools were kept separate, and other pools in which both target species were represented were finally pooled. [Figure 11] Binding of the CH3C clone identified from the initial soft-randomized library to human and cynomolgus monkey TfR is shown. Positive controls were the high-affinity anti-TfR antibody Ab204 and the low-affinity anti-TfR antibody Ab084. (A) Binding to human TfR is shown. (B) Binding to cynomolgus monkey TfR is shown. [Figure 12] Figure 1 shows binding of CH3C clones identified from the initial soft-randomized library to human TfR in the presence or absence of holo-Tf. Clones were in Fc-Fab fusion format. Ab204, a high-affinity anti-TfR antibody, was used as a positive control for this assay. (A) Binding of CH3C mutants to human TfR coated on ELISA plates is shown. (B) Binding of CH3C mutants to human TfR coated on ELISA plates in the presence of 5 μM holo-Tf. [Figure 13] Figure 1 shows binding of CH3C clones identified from the initial soft-randomized library to 293F cells. Cells were distributed into 96-well V-bottom plates, and different concentrations of CH3C clones formatted as Fc-Fab fusion proteins were added. After 1 hour of incubation at 4°C, each plate was centrifuged, washed, and then incubated with goat anti-human IgG-Alexa Fluor® 647 secondary antibody for 30 minutes at 4°C. After further washing of the cells, each plate was read on a FACSCanto™ II flow cytometer, and median fluorescence in the APC (647 nm) channel was determined using FlowJo® software. [Figure 14]Figure 1 shows binding of CH3C clones identified from the initial soft-randomized library to CHO-K1 cells. Cells were distributed into 96-well V-bottom plates and different concentrations of CH3C clones formatted as Fc-Fab fusions were added. After 1 hour of incubation at 4°C, each plate was centrifuged, washed, and then incubated with goat anti-human IgG-Alexa Fluor® 647 secondary antibody for 30 minutes at 4°C. After further washing of the cells, each plate was read on a FACSCanto™ II flow cytometer, and median fluorescence in the APC (647 nm) channel was determined using FlowJo® software. (A) CHO-K1 cells overexpressing human TfR are shown. (B) CHO-K1 cells overexpressing cynomolgus monkey TfR are shown. (C) CHO-K1 parental cells not expressing human TfR are shown. [Figure 15] The apical domain of the TfR is shown. (A) The location of the apical domain of the human TfR protein is shown. The inset shows a close-up of the seven residues that differ between human and cynomolgus TfR. (B) A sequence alignment including the seven residues that differ between human TfR (SEQ ID NO:107) and cynomolgus TfR (SEQ ID NO:108) is shown. The consensus sequence is SEQ ID NO:622. [Figure 16]Binding of CH3C clones to phage-displayed apical domains is shown. (A) Myc expression of different TfR apical domain mutants is shown, demonstrating similar and normalized expression levels for each mutant. (B) Binding of CH3C.18 to wild-type and mutant human TfR apical domains is shown, demonstrating low binding to the R208G mutant. (C) Binding of CH3C.35 to wild-type and mutant human TfR apical domains is shown, demonstrating low binding to the R208G mutant. (D) Binding of CH3C.18 to wild-type human and cynomolgus monkey TfR apical domains and the G208R mutant cynomolgus monkey apical domain is shown, demonstrating restoration of binding to the mutants. (E) Binding of CH3C.35 to wild-type human and cynomolgus monkey TfR apical domains and the G208R mutant cynomolgus monkey apical domain is shown, demonstrating restoration of binding to the mutants. [Figure 17A] Paratope mapping of CH3C mutants by reverting the mutated position to the wild-type residue is shown. (A) Paratope mapping of CH3C.35 by ELISA binding to human TfR for revertants. (B) Paratope mapping of CH3C.35 by ELISA binding to cynomolgus monkey TfR for revertants. (C) Paratope mapping of CH3C.18 by ELISA binding to human TfR for revertants. (D) Paratope mapping of CH3C.18 by ELISA binding to cynomolgus monkey TfR for revertants. [Figure 17B] See legend to Figure 17A. [Figure 17C] See legend to Figure 17A. [Figure 17D] See legend to Figure 17A. [Figure 18] Design of a consensus mutation library for CH3C. (A) A consensus library based on a CH3C.35-like sequence. (B) A consensus library based on a CH3C.18-like sequence. (C) A gapped library based on CH3C.18 and CH3C.35. (D) An aromatic library based on CH3C.18. [Figure 19] Figure 1 shows binding ELISA of CH3C variants from the consensus mutation library to human or cynomolgus monkey TfR. While the new variants (i.e., CH3C.3.2-1, CH3C.3.2-5, and CH3C.3.2-19) had similar binding EC50 values ​​to cynomolgus monkey and human TfR, the parent clones CH3C.18 and CH3C.35 had significantly higher EC50 values ​​to human TfR versus cynomolgus monkey TfR. (A) Data for CH3C.3.2-1 is shown. (B) Data for CH3C.3.2-19 is shown. (C) Data for CH3C.3.2-5 is shown. (D) Data for CH3C.18 is shown. (E) Data for CH3C.35 is shown. [Figure 20] Internalization of CH3C variants from the consensus maturation library in human (HEK293) and monkey (LLC-MK2) cells is shown. Clones CH3C.3.2-5 and CH3C3.2-19, which have similar affinities for human and cynomolgus monkey TfR, showed significantly higher uptake into monkey cells compared with clone CH3C.35, which bound better to human TfR. Anti-BACE1 antibody Ab107 was used as a negative control (BACE1 is not expressed in either HEK293 or MK2 cells). Anti-TfR antibody Ab204 was used as a positive control. [Figure 21] A map of the NNK walk residues shown on the CH structure is shown (source: PDB 4W4O). The black surface shows the original CHC register, the gray surface shows the 44 residues incorporated into the NNK walk structure, and the ribbon shows the wild-type backbone. [Figure 22] Enriched yeast populations after three rounds of sorting of the NNKwalk library are shown. Yeast were stained with anti-c-Myc and monitored for expression (x-axis) and binding (y-axis) to the TfR apical domain (200 nM cynomolgus monkey or 200 nM human). The data shown here clearly demonstrate high binding to both orthologs of the TfR apical domain. [Figure 23A]FACS data for CH3C.35.21 mutants are shown. Yeast were stained with anti-c-Myc and monitored for expression (x-axis) and binding (y-axis) to the human TfR apical domain (200 nM). FACS data for clone CH3C.35.21 are shown. [Figure 23B] FACS data are shown for CH3C.35.21 mutants. Clone CH3C.35.21 was either reverted to wild type at 11 positions (top row of FACS plots) or expressed as an NNK library of all 20 amino acids (bottom row of FACS plots, before sorting). [Figure 24A] ELISA comparison of bivalent and monovalent CH3C polypeptides binding to human and cynomolgus monkey TfR. (A) Bivalent CH3C polypeptide binding to human TfR. (B) Bivalent CH3C polypeptide binding to cynomolgus monkey TfR. (C) Monovalent CH3C polypeptide binding to human TfR. (D) Monovalent CH3C polypeptide binding to cynomolgus monkey TfR. [Figure 24B] See legend to Figure 24A. [Figure 24C] See legend to Figure 24A. [Figure 24D] See legend to Figure 24A. [Figure 25A] Cell binding of monovalent CH3C polypeptides is shown. (A) 293 cells are shown. (B) A magnified view of binding to the 293 cells shown in (A). (C) CHO-K1 cells stably transfected with human TfR are shown. (D) A magnified view of binding to CHO-K1 cells stably transfected with human TfR are shown in (C). (E) CHO-K1 cells stably transfected with cynomolgus monkey TfR are shown. [Figure 25B] See legend to Figure 25A. [Figure 25C] See legend to Figure 25A. [Figure 25D] See legend to Figure 25A. [Figure 25E] See legend to Figure 25A. [Figure 26] 1 shows the internalization of monovalent and bivalent CH3C polypeptides in HEK293 cells. [Figure 27-1] Binding kinetics of CH3C polypeptides are shown. (A) Binding data for CH3C.35.N163 to human TfR is shown. (B) Binding data for CHC3.35 to human TfR is shown. (C) Monovalent binding data for CHC3.35.N163 binding to human TfR is shown. (D) Monovalent binding data for CHC3.35 to human TfR is shown. (E) Binding data for CH3C.35.N163 to cynomolgus monkey TfR is shown. (F) Binding data for CHC3.35 to cynomolgus monkey TfR is shown. (G) Monovalent binding data for CHC3.35.N163 binding to cynomolgus monkey TfR is shown. (H) Monovalent binding data for CHC3.35 to cynomolgus monkey TfR is shown. [Figure 27-2] See description of Figure 27-1. [Figure 28-1] Binding kinetics of CH3C polypeptides are shown. (A) Binding data for CH3C.3.2-1 to human TfR is shown. (B) Binding data for CH3C.3.2-5 to human TfR is shown. (C) Binding data for CH3C.3.2-19 to human TfR is shown. (D) Binding data for CH3C.3.2-1 to cynomolgus monkey TfR is shown. (E) Binding data for CH3C.3.2-5 to cynomolgus monkey TfR is shown. (F) Binding data for CH3C.3.2-19 to cynomolgus monkey TfR is shown. [Figure 28-2] See description of Figure 28-1. [Figure 29A] Binding of polypeptide-Fab fusions to FcRn at pH 5.5 in the presence (lower trace) or absence (upper trace) of the human TfR extracellular domain is shown. Data for clone CH3C.35 are shown (FIG. 29A). [Figure 29B] Binding of polypeptide-Fab fusions to FcRn at pH 5.5 in the presence (lower trace) or absence (upper trace) of the human TfR extracellular domain is shown. Data is shown for clone CH3C.35.19. [Figure 29C]Binding of polypeptide-Fab fusions to FcRn at pH 5.5 in the presence (lower trace) or absence (upper trace) of the human TfR extracellular domain is shown. Data is shown for clone CH3C.35.20. [Figure 29D] Binding of polypeptide-Fab fusions to FcRn at pH 5.5 in the presence (lower trace) or absence (upper trace) of the human TfR extracellular domain is shown. Data is shown for clone CH3C.35.21. [Figure 29E] Binding of polypeptide-Fab fusions to FcRn at pH 5.5 in the presence (lower trace) or absence (upper trace) of the human TfR extracellular domain is shown. Data is shown for clone CH3C.35.24. [Figure 30] Pharmacokinetic (PK) analysis of CH3C polypeptides in wild-type mice shows that all polypeptide-Fab fusions exhibited comparable clearance to wild-type Fc-Fab fusions (i.e., anti-RSV antibody Ab122 and anti-BACE1 antibody Ab153), except for CH3C.3.2-5, which exhibited faster clearance. [Figure 31] Brain pharmacokinetic / pharmacodynamic (PK / PD) data are shown for mouse brain tissue. Chimeric huTfR heterozygous mice (n=4 / group) were intravenously administered 42 mg / kg of either Ab153 or monovalent CH3C.35.N163 (denoted as "CH3C.35.N163_monovalent"), and wild-type mice (n=3) were intravenously administered 50 mg / kg of control human IgG1 (denoted as "huIgG1"). Bars represent the mean ± SD. [Figure 32] IgG concentrations in hTfR apical + / + mice 24 hours after treatment with 50 mg / kg of polypeptide are shown. (A) IgG concentrations in plasma are shown. (B) IgG concentrations in brain tissue are shown. [Figure 33] Figure 1 shows target binding of polypeptides administered to hTfR apical + / + mice after 24 hours, as measured by reduction in amyloid beta protein 50 (Aβ40). (A) Aβ40 concentrations in plasma are shown. (B) Aβ40 concentrations in brain tissue are shown. [Figure 34] SDS-PAGE gel of size fractionation of the complex of CH3C.3.18Fc and the TfR apical domain (AD). Lane 1: molecular weight marker. Lane 2: reduced CH3C.18Fc-AD complex after size exclusion chromatography. [Figure 35A] 1 shows the binding of the polypeptide of the present invention to the transferrin receptor, and the binding interface between clone CH3C.18 and the apical domain of the transferrin receptor. [Figure 35B] FIG. 35B shows an enlarged view of the bonding surface shown in FIG. 35A. [Figure 36]The interaction of CH3C.18 with the TfR apical domain is shown. (A) The structural architecture of the TfR apical domain and CH3C.18Fc (top) and the binding surface (within 5 Å) of the TfR apical domain and CH3C.18Fc (bottom) are shown. The co-complex structure was resolved at 3.6 Å resolution. This structure reveals the epitope of the TfR apical domain bound to CH3C.18. Specifically, the N-terminal region of the apical domain is involved in CH3C Fc binding, and the structure is consistent with the mutagenesis data of CH3C.18Fc and the TfR apical domain. Furthermore, all side chains of the CH3C.18 library make contact with TfR (within 5 Å). Residues in the CH3C.18 library: L157, H159, V160, W161, A162, V163, P186, T189, and W194. Non-library residues: F196 and S156. (B) The main interactions between CH3C.18Fc and the TfR apical domain are shown. The cation-π interaction between W161 on CH3C.18Fc and R208 on the apical domain is the central binding interaction. Mutation of W388 in CH3C.18 or R208 in the apical domain disrupts binding of CH3C.18Fc to the apical domain. Consistent with this, the F208G mutation from human to cynomolgus monkey explains the lower affinity in cynomolgus monkeys. Furthermore, residues not conserved in the human apical domain (N292 and E294, respectively; K292 and D294 in cynomolgus monkeys) are nearby. Therefore, mutating Q192 in CH3C.18Fc can selectively enhance cynomolgus binding relative to human binding. [Figure 37A] 1 shows the binding of a polypeptide of the invention to the transferrin receptor, showing hydrogen bond and non-bond contacts between residues of clone CH3C.18 (A chain) and residues of the apical domain of the transferrin receptor (D chain). [Figure 37B] 1 shows the binding of a polypeptide of the invention to the transferrin receptor, showing hydrogen bond and non-bond contacts between residues of clone CH3C.18 (B strand) and residues of the apical domain of the transferrin receptor (C strand). [Figure 38]1 shows an alignment of the amino acid sequences of human IgG1, IgG2, IgG3, and IgG4 (SEQ ID NOs: 623 to 626). [Figure 39] Figure 1 shows the binding of a polypeptide of the present invention to the transferrin receptor. (A) The structural architecture of the TfR apical domain and CH3C.35Fc (top) and the binding surface (within 5 Å) of the TfR apical domain and CH3C.35Fc (bottom) are shown. The co-complex structure was resolved at a resolution of 3.4 Å. In this structure, the epitope of the TfR apical domain bound to CH3C.35 is visible. All side chains of the CH3C.35 library are in contact with TfR (within 5 Å). CH3C.35 library residues: Y157, T159, E160, W161, S162, T186, E189, and W194. Non-library residues: F196, S156, Q192. (B) and (C) show enlarged views of the binding interaction between clone CH3C.35 shown in (A) and the apical domain of the transferrin receptor. [Figure 40] (A) Superimposed structure of the complex between CH3C.35Fc and TfR-AD and the complex between CH3C.18Fc and TfR-AD. (B) Enlarged view of the superimposed structure in (A). [Figure 41A] 1 shows the binding of a polypeptide of the invention to the transferrin receptor, showing hydrogen bond and non-bond contacts between residues of clone CH3C.35 (A chain) and residues of the apical domain of the transferrin receptor (D chain). [Figure 41B] 1 shows the binding of a polypeptide of the invention to the transferrin receptor, showing hydrogen bond and non-bond contacts between residues of clone CH3C.35 (B strand) and residues of the apical domain of the transferrin receptor (C strand). [Figure 42]Figure 1 shows plasma PK and Aβ40 reduction in cynomolgus monkeys for Fc-Fab fusion polypeptides containing the CH3C variant fused to the Fab domain of Ab153. (A) Ab210 and CH3C.35.9:Ab153 showed faster clearance via TfR-mediated clearance compared to control IgG (Ab122) and Ab153. (B) Ab153, Ab210, and CH3C.35.9:Ab153, all of which bind to and inhibit BACE1, showed significant Aβ40 reduction in plasma. [Figure 43] Figure 1 shows significant reductions in cerebrospinal fluid (CSF) Aβ and sAPPβ / sAPPα by Fc-Fab fusion polypeptides containing the CH3C variant fused to the Fab domain of Ab153 in cynomolgus monkeys. (A) Animals treated with Ab210 and CH3C.35.9:Ab153 showed a 70% reduction in Aβ40 in CSF compared to Ab153 and control IgG (Ab122). (B) Animals treated with Ab210 and CH3C.35.9:Ab153 showed a 75% reduction in the sAPPβ / sAPPα ratio compared to Ab153 and control IgG (Ab122). n = 4 / group. Line graphs represent the mean ± SEM. [Figure 44A](A) and (B) show huIgG1 concentrations in plasma (A) and brain lysates (B) of hTfR apical + / + knock-in (KI) mice after a single 50 mg / kg systemic injection of anti-BACE1_Ab153, CH3C35.21:Ab153, CH3C35.20:Ab153, or CH3C35:Ab153 polypeptide fusions (mean ± SEM, n = 5 / group). (C) shows the concentration of endogenous mouse Aβ in brain lysates of hTfR apical + / + KI mice after a single 50 mg / kg systemic injection of anti-BACE1_Ab153, CH3C35.21:Ab153, CH3C35.20:Ab153, or CH3C35:Ab153 polypeptide fusions (mean ± SEM, n = 5 / group). (D) shows quantification by Western blot of brain TfR protein normalized to actin in brain lysates of hTfR apical + / + KI mice after a single 50 mg / kg systemic injection of anti-BACE1_Ab153, CH3C35.21:Ab153, CH3C35.20:Ab153, or CH3C35:Ab153 polypeptide fusions (mean ± SEM, n = 5 / group). [Figure 44B] See legend to Figure 44A. [Figure 44C] See legend to Figure 44A. [Figure 44D] See legend to Figure 44A. [Figure 45A](A) and (B) show huIgG1 concentrations in plasma (A) and brain lysates (B) of hTfR apical + / + KI mice after a single 50 mg / kg systemic injection of anti-BACE1_Ab153, CH3C.35.23:Ab153, or CH3C.35.23.3:Ab153 polypeptide fusions (mean ± SEM, n = 5 / group). (C) shows the concentration of endogenous mouse Aβ in brain lysates of hTfR apical + / + KI mice after a single 50 mg / kg systemic injection of anti-BACE1_Ab153, CH3C.35.23:Ab153, or CH3C.35.23.3:Ab153 polypeptide fusions (mean ± SEM, n = 5 / group). (D) shows quantification by Western blot of brain TfR protein normalized to actin in brain lysates of hTfR apical + / + KI mice after a single 50 mg / kg systemic injection of anti-BACE1_Ab153, CH3C.35.23:Ab153, or CH3C.35.23.3:Ab153 polypeptide fusions (mean ± SEM, n = 4 / group). [Figure 45B] See legend to Figure 45A. [Figure 45C] See legend to Figure 45A. [Figure 45D] See legend to Figure 45A. [Figure 46A] Figure 1 shows a 28-day PKPD study in cynomolgus monkeys after administration of a single 30 mg / kg dose of the indicated protein. (A) and (B) show serum huIgG1 and plasma Aβ concentrations in plasma, demonstrating the peripheral exposure of the administered compound over time and the resulting effect on plasma Aβ concentrations. (C) and (D) show Aβ and sAPPβ / sAPPα in the CSF of cynomolgus monkeys after administration (mean ± SEM, n = 4-5 per group). [Figure 46B] See legend to Figure 46A. [Figure 46C] See legend to Figure 46A. [Figure 46D] See legend to Figure 46A. [Figure 47]Blood reticulocytes (A), absolute serum iron concentration (B), and absolute red blood cell counts (C) relative to pre-dose concentrations are shown in peripheral blood in cynomolgus monkeys after administration of a single 30 mg / kg dose of each indicated protein (mean ± SEM, n = 4–5 / group). [Figure 48] Peripheral PK analysis (plasma huIgG1 concentrations (A) and clearance values ​​(B)) of each indicated protein in hFcRn knock-in mice after a single 10 mg / kg intravenous injection over 14 days is shown (mean ± SEM, n = 3 / group). [Figure 49] Median fluorescence intensities of TfR-bound CH3C.18 mutants are shown. DETAILED DESCRIPTION OF THE INVENTION

[0104] Detailed Description of the Invention I. Introduction Disclosed herein are polypeptides that bind to the transferrin receptor (TfR). The invention is based, in part, on the discovery that modifying specific amino acids within the Fc region can generate novel binding sites within Fc polypeptides specific for TfR. Taking advantage of the fact that TfR is highly expressed at the blood-brain barrier (BBB) ​​and that TfR naturally translocates transferrin from the blood to the brain, these polypeptides can be used to transport therapeutic agents (e.g., therapeutic polypeptides, antibody variable regions such as Fab, and small molecules) across the BBB. This approach can significantly improve brain uptake of therapeutic agents and is therefore extremely useful for treating disorders and diseases where delivery to the brain is beneficial.

[0105] In one aspect, the invention is based in part on the discovery that polypeptides that bind to the transferrin receptor can be generated by substituting specific sets of amino acids within a CH3 or CH2 domain polypeptide. Accordingly, in one aspect, provided herein are transferrin receptor binding polypeptides having multiple substitutions in the following sets of amino acids: (i) 157, 159, 160, 161, 162, 163, 186, 189, and 194, or (ii) 118, 119, 120, 122, 210, 211, 212, and 213, numbered relative to SEQ ID NO:1. In some embodiments, transferrin receptor binding polypeptides of the invention have multiple substitutions at amino acid positions (iii) 47, 49, 56, 58, 59, 60, 61, 62, and 63; (iv) 39, 40, 41, 42, 43, 44, 68, 70, 71, and 72; (v) 41, 42, 43, 44, 45, 65, 66, 67, 69, and 73; or (vi) 45, 47, 49, 95, 97, 99, 102, 103, and 104, numbered relative to SEQ ID NO:1. Any four to all of the amino acid positions within a set can be substituted. For purposes of this disclosure, substitutions are determined relative to SEQ ID NO:1. Thus, an amino acid is considered a substitution if it is different from the amino acid at the corresponding position in SEQ ID NO:1, even if that amino acid occurs at that position in a naturally occurring CH3 or CH2 domain polypeptide.

[0106] Also provided herein are methods for producing transferrin receptor binding polypeptides by generating mutant polypeptides having substitutions at positions in set (i), (ii), (iii), (iv), (v), or (vi). Such mutants can be analyzed for transferrin receptor binding as described herein and further mutated to enhance binding.

[0107] In a further aspect, provided herein are methods of treatment and methods of using transferrin receptor binding polypeptides that target compositions to transferrin receptor-expressing cells (e.g., deliver the compositions to the cells or deliver the compositions across an endothelium, such as the blood-brain barrier).

[0108] II. Definition As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "polypeptide" may include two or more such molecules.

[0109] As used herein, the terms "about" and "approximately," when used to modify a quantity specified in a numerical value or range, indicate that the numerical value and reasonable deviations from that value known to those of ordinary skill in the art, such as ±20%, ±10%, or ±5%, are within the expected meaning of the stated value.

[0110] As used in the context of the present invention, "transferrin receptor" or "TfR" refers to transferrin receptor protein 1. The polypeptide sequence of human transferrin receptor 1 is set forth in SEQ ID NO:235. Transferrin receptor protein 1 sequences from other species are also known (e.g., chimpanzee (accession number XP_003310238.1), rhesus monkey (NP_001244232.1), dog (NP_001003111.1), cow (NP_001193506.1), mouse (NP_035768.1), rat (NP_073203.1), and chicken (NP_990587.1)). The term "transferrin receptor" also encompasses allelic variants of an exemplary reference sequence (e.g., a human sequence) encoded by a gene at the chromosomal locus of transferrin receptor protein 1. The full-length transferrin receptor protein contains a short N-terminal intracellular region, a transmembrane region, and a large extracellular domain. The extracellular domain is characterized by three domains: a protease-like domain, a helical domain, and an apical domain. The apical domain sequence of human transferrin receptor 1 is set forth in SEQ ID NO:107.

[0111] As used herein, the terms "CH3 domain" and "CH2 domain" refer to immunoglobulin constant region domain polypeptides. In the context of an IgG antibody, a CH3 domain polypeptide refers to the segment of amino acids from about 341 to about 447 when numbered according to the EU numbering scheme, and a CH2 domain polypeptide refers to the segment of amino acids from about 231 to about 340 when numbered according to the EU numbering scheme. CH2 and CH3 domain polypeptides may also be numbered according to the IMGT (ImMunoGeneTics) numbering scheme, in which the CH2 domain is numbered 1 to 110 and the CH3 domain is numbered 1 to 107 according to the IMGT Scientific chart numbering (IMGT website). The CH2 and CH3 domains are part of the Fc region of an immunoglobulin. In the context of an IgG antibody, an Fc region refers to the segment of amino acids from about 231 to about 447 when numbered according to the EU numbering scheme. As used herein, the term "Fc region" may also include at least a portion of an antibody hinge region. An exemplary hinge region sequence is set forth in SEQ ID NO:234.

[0112] The terms "wild-type," "native," and "naturally occurring" with respect to CH3 and CH2 domains are used herein to refer to domains having sequences that occur in nature.

[0113] In the context of the present invention, the term "mutant" is used interchangeably with "variant" in reference to a mutant polypeptide or mutant polynucleotide. With respect to a given wild-type CH3 or CH2 domain reference sequence, variants can include naturally occurring allelic variants. A "non-naturally occurring" CH3 or CH2 domain refers to a variant or mutant domain that does not naturally occur in cells and is generated by genetic modification (e.g., using genetic engineering techniques or mutagenesis) of a native CH3 or CH2 domain polynucleotide or polypeptide. A "variant" includes any domain that contains at least one amino acid mutation relative to the wild-type. Mutations can include substitutions, insertions, and deletions.

[0114] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.

[0115] Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. "Amino acid analogs" refer to compounds (e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium, etc.) that have the same basic chemical structure as a naturally occurring amino acid (i.e., an α carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group). Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. "Amino acid mimetics" refer to compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0116] Naturally occurring α-amino acids include, but are not limited to, alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Naturally occurring stereoisomers of α-amino acids include, but are not limited to, D-alanine (D-Ala), D-cysteine ​​(D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), and D-lysine. (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

[0117] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0118] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. Such terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers. An amino acid polymer can be entirely L-amino acids, entirely D-amino acids, or a mixture of L- and D-amino acids.

[0119] The terms "conservative substitution," "conservative mutation," or "conservatively modified variant" refer to a change that results in the substitution of one amino acid with another amino acid that can be classified as having similar properties. Examples of categories of conservative amino acid groups defined in this manner include "charged / polar groups" including Glu (glutamic acid or E), Asp (aspartic acid or D), Asn (asparagine or N), Gln (glutamine or Q), Lys (lysine or K), Arg (arginine or R), and His (histidine or H); "aromatic groups" including Phe (phenylalanine or F), Tyr (tyrosine or Y), Trp (tryptophan or W), and (histidine or H); and "aliphatic groups" including Gly (glycine or G), Ala (alanine or A), Val (valine or V), Leu (leucine or L), Ile (isoleucine or I), Met (methionine or M), Ser (serine or S), Thr (threonine or T), and Cys (cysteine ​​or C). Subgroups may also be identified within each group. For example, the group of charged or polar amino acids can be further divided into subgroups including a "positively charged subgroup" consisting of Lys, Arg, and His, a "negatively charged subgroup" consisting of Glu and Asp, and a "polar subgroup" consisting of Asn and Gln. In another example, aromatic or cyclic groups can be further divided into subgroups including a "nitrogen ring subgroup" consisting of Pro, His, and Trp, and a "phenyl subgroup" consisting of Phe and Tyr. In yet another example, aliphatic groups can be further divided into subgroups such as an "aliphatic non-polar subgroup" consisting of Val, Leu, Gly, and Ala, and an "aliphatic weakly polar subgroup" consisting of Met, Ser, Thr, and Cys.Examples of conservative mutation categories include amino acid substitutions of amino acids within the above subgroups, such as, but not limited to, Lys for Arg, or vice versa, to maintain a positive charge; Glu for Asp, or vice versa, to maintain a negative charge; Ser for Thr, or vice versa, to maintain a free -OH; Gln for Asn, or vice versa, to maintain a free -NH. In some embodiments, hydrophobicity is maintained, for example, by substituting a hydrophobic amino acid for a naturally occurring hydrophobic amino acid in the active site.

[0120] The terms "identical" or "identity" in the context of two or more polypeptide sequences refer to two or more sequences or subsequences that are the same over a specified region when compared and aligned for maximum correspondence over a comparison window, or designated region, as determined using a sequence comparison algorithm or by manual alignment and visual inspection, or that have a specified percentage (%) of amino acid residues that are identical, for example, at least 60% identical, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical or more.

[0121] In polypeptide sequence comparison, one amino acid sequence generally serves as a reference sequence to which candidate sequences are compared. Alignment can be performed using various methods available to those skilled in the art to achieve maximum alignment, such as visual alignment or using publicly available software with known algorithms. Such programs include the BLAST program, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.), or Megalign (DNASTAR). The parameters used in alignment to achieve maximum alignment can be determined by those skilled in the art. For the purposes of this application, the standard protein BLAST of the BLASTP algorithm is used to align two protein sequences using default parameters.

[0122] The terms "corresponding to," "determined with respect to," or "numbered with respect to," when used in the context of identifying a given amino acid residue in a polypeptide sequence, refer to the position of the residue in a particular reference sequence when the given amino acid sequence is compared to the reference sequence for maximum alignment. Thus, for example, an amino acid residue in a polypeptide "corresponds" to an amino acid in the region from amino acid 114 to amino acid 220 of SEQ ID NO:1 when the residue is aligned with that amino acid in SEQ ID NO:1 when optimally aligned with SEQ ID NO:1. A polypeptide that is aligned with a reference sequence need not be the same length as the reference sequence.

[0123] As used herein, "binding affinity" refers to the strength of a non-covalent interaction between two molecules, e.g., between one binding site of a polypeptide and a target to which the polypeptide binds, e.g., the transferrin receptor. Thus, for example, the term may refer to a 1:1 interaction between a polypeptide and its target unless otherwise indicated or clear from the context. Binding affinity is measured by the dissociation rate constant (k d ,time -1) as the binding rate constant (k a ,time -1 M -1 ) is the equilibrium dissociation constant (K D ) can be quantified by measuring the K D can be determined by measuring the kinetics of complex formation and dissociation using, for example, surface plasmon resonance (SPR) methods such as the Biacore™ system; kinetic exclusion assays such as KinExA®; and biolayer interferometry (e.g., using the ForteBio® Octet® platform). As used herein, "binding affinity" includes not only formal binding affinity, such as that reflecting a 1:1 interaction between a polypeptide and its target, but also K values, which may reflect strong binding. D The apparent affinity calculated is also included.

[0124] The phrases "specifically bind" or "selectively bind" to a target, e.g., transferrin receptor, when referring to a polypeptide comprising a modified CH3 and / or modified CH2 domain described herein, refer to a binding reaction in which the polypeptide binds to the target with greater affinity, greater avidity, and / or longer duration than it binds to a structurally different target, e.g., a target that is not a member of the transferrin receptor family. In typical embodiments, the polypeptide has at least 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold, or 100-fold or greater affinity for the transferrin receptor compared to an unrelated target when assayed under the same affinity assay conditions. In some embodiments, the modified CH3 and / or modified CH2 domain polypeptide specifically binds to an epitope on the transferrin receptor that is conserved between species, e.g., between non-human primates and human species. In some embodiments, the polypeptide can bind only to the human transferrin receptor.

[0125] The terms "subject," "individual," and "patient," used interchangeably herein, refer to mammals, including, but not limited to, humans, non-human primates, rodents (e.g., rats, mice, and guinea pigs), rabbits, cows, pigs, horses, and other mammalian species. In one embodiment, the patient is a human.

[0126] The terms "treatment," "treating," and similar terms are used herein generally to mean obtaining a desired pharmacological and / or physiological effect. "Treating" or "treatment" can refer to any indication of success in treating or ameliorating an injury, disease, or condition, including any objective or subjective parameter, such as relief, remission, improved patient survival, increased survival time or rate, disappearance of symptoms or making the injury, disease, or condition more tolerable to the patient, slowing the rate of degeneration or decline, or improvement in the patient's physical or mental health. Treatment or amelioration of symptoms can be based on objective or subjective parameters. The effect of treatment can be compared to an individual or population of individuals not receiving treatment, or to the same patient before or at different time points during treatment.

[0127] "Pharmaceutically acceptable excipient" refers to a non-active pharmaceutical ingredient that is biologically or pharmacologically compatible for use in humans or animals, such as, but not limited to, a buffer, carrier, or preservative.

[0128] As used herein, a "therapeutic amount" or "therapeutically effective amount" of a drug is an amount of drug that treats, alleviates, relieves, or reduces the severity of symptoms of a disease in a subject. A "therapeutic amount" or "therapeutically effective amount" of a drug can improve a patient's survival, increase the length or rate of survival, eliminate symptoms, make an injury, disease, or condition more tolerable, slow the rate of degeneration or decline, or improve the patient's physical or mental well-being.

[0129] The term "administering" refers to a method of delivering an agent, compound, or composition to a site where a biological effect is desired. These methods include, but are not limited to, topical, parenteral, intravenous, intradermal, intramuscular, intrathecal, colonic, rectal, or intraperitoneal administration. In one embodiment, the polypeptides described herein are administered intravenously.

[0130] III. Transferrin Receptor Binding Polypeptides This section describes the production of polypeptides according to the invention that are capable of binding to the transferrin receptor and being transported across the blood-brain barrier (BBB).

[0131] In one aspect, a polypeptide is provided that includes a CH3 or CH2 domain with modifications that enable the polypeptide to specifically bind to the transferrin receptor. Such modifications are introduced into a specific set of amino acids present on the surface of the CH3 or CH2 domain. In some embodiments, the polypeptide that includes the modified CH3 or CH2 domain specifically binds to an epitope within the apical domain of the transferrin receptor.

[0132] Those skilled in the art will appreciate that the CH2 and CH3 domains of other immunoglobulin isotypes, such as IgM, IgA, IgE, and IgD, can be similarly modified by identifying amino acids within these domains that correspond to the above-described sets (i) through (vi). Modifications can also be made to corresponding domains from immunoglobulins derived from other species, such as non-human primates, monkeys, mice, rats, rabbits, dogs, pigs, and chickens.

[0133] CH3 Transferrin Receptor Binding Polypeptide In some embodiments, the domain to be modified is a human Ig CH3 domain, such as an IgG CH3 domain. The CH3 domain may be derived from any IgG subtype, i.e., IgG1, IgG2, IgG3, or IgG4. In the context of IgG antibodies, the CH3 domain refers to the segment of amino acids from about 341 to about 447 when numbered according to the EU numbering scheme. Positions within the CH3 domain for purposes of identifying a set of corresponding amino acid positions for transferrin receptor binding are determined relative to SEQ ID NO:3 or relative to amino acids 114-220 of SEQ ID NO:1, unless otherwise specified. Substitutions are also determined relative to SEQ ID NO:1, i.e., an amino acid is considered a substitution for the amino acid at the corresponding position in SEQ ID NO:1. SEQ ID NO:1 contains a partial hinge region sequence, PCP, as amino acids 1-3. The numbering of positions within the CH3 domain relative to SEQ ID NO:1 includes these first three amino acids.

[0134] As noted above, sets of residues in a CH3 domain that can be modified in accordance with the present invention are numbered herein with reference to SEQ ID NO:1. For example, any CH3 domain, such as the CH3 domain of IgG1, IgG2, IgG3, or IgG4, can have modifications (e.g., amino acid substitutions) at one or more sets of residues that correspond to the residues at the recited positions in SEQ ID NO:1. An alignment of the amino acid sequence of human IgG1 of SEQ ID NO:1 with human IgG2, IgG3, and IgG4 is shown in Figure 38. The respective positions in the IgG2, IgG3, and IgG4 sequences that correspond to any particular position in SEQ ID NO:1 can be readily determined.

[0135] In one embodiment, the modified CH3 domain polypeptide that specifically binds to the transferrin receptor binds to the apical domain of the transferrin receptor at an epitope that includes position 208 of the full-length human transferrin receptor sequence (SEQ ID NO:235), which corresponds to position 11 of the apical domain sequence of the human transferrin receptor set forth in SEQ ID NO:107. SEQ ID NO:107 corresponds to amino acids 198-378 of the human transferrin receptor 1 uniprotein sequence P02786 (SEQ ID NO:235). In some embodiments, the modified CH3 domain polypeptide binds to the apical domain of the transferrin receptor at an epitope including positions 158, 188, 199, 207, 208, 209, 210, 211, 212, 213, 214, 215, and / or 294 of the full-length human transferrin receptor sequence (SEQ ID NO:235). The modified CH3 domain polypeptide may bind to the transferrin receptor without blocking or otherwise inhibiting the binding of transferrin to the receptor. In some embodiments, the binding of transferrin to the TfR is not substantially inhibited. In some embodiments, the binding of transferrin to the TfR is inhibited by less than about 50% (e.g., less than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%). In some embodiments, binding of transferrin to TfR is inhibited by less than about 20% (e.g., less than about 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%). Exemplary CH3 domain polypeptides that exhibit such binding specificity include polypeptides with amino acid substitutions at positions 157, 159, 160, 161, 162, 163, 186, 189, and 194, relative to amino acids 114-220 of SEQ ID NO:1.

[0136] Group (i) that binds to the transferrin receptor of CH3: 157, 159, 160, 161, 162, 163, 186, 189, and 194 In some embodiments, modified CH3 domain polypeptides of the invention contain at least three or at least four, and typically five, six, seven, eight, or nine, substitutions at a set of amino acid positions including 157, 159, 160, 161, 162, 163, 186, 189, and 194 (set i). Exemplary substitutions that can be introduced at these positions are shown in Table 6. In some embodiments, the amino acid at position 161 and / or 194 is an aromatic amino acid (e.g., Trp, Phe, or Tyr). In some embodiments, the amino acid at position 161 is Trp. In some embodiments, the amino acid at position 161 is Gly. In some embodiments, the aromatic amino acid at position 194 is Trp or Phe.

[0137] In some embodiments, modified CH3 domain polypeptides that specifically bind to the transferrin receptor comprise at least one position having a substitution relative to SEQ ID NO:1, as follows: Leu, Tyr, Met, or Val at position 157; Leu, Thr, His, or Pro at position 159; Val, Pro, or an acidic amino acid at position 160; an aromatic amino acid, such as Trp or Gly (e.g., Trp), at position 161; Val, Ser, or Ala at position 162; an acidic amino acid, Ala, Ser, Leu, Thr, or Pro at position 186; Thr or an acidic amino acid at position 189; or Trp, Tyr, His, or Phe at position 194. In some embodiments, modified CH3 domain polypeptides can include conservative substitutions of particular amino acids at one or more positions within the above sets, e.g., amino acids within groups based on the same charge, hydrophobicity, side chain ring structure (e.g., aromatic amino acids), size, and / or polar or nonpolar. Thus, for example, Ile may be present at position 157, 159, and / or 186. In some embodiments, the acidic amino acid at one, two, or each of positions 160, 186, and 189 is Glu. In other embodiments, the acidic amino acid at one, two, or each of positions 160, 186, and 189 is Asp. In some embodiments, two, three, four, five, six, seven, or all eight of positions 157, 159, 160, 161, 162, 186, 189, and 194 have an amino acid substitution as specified in this paragraph.

[0138] In some embodiments, a CH3 domain polypeptide having modifications in set (i) comprises a native Asn at position 163. In some embodiments, the modified CH3 domain polypeptide comprises Gly, His, Gln, Leu, Lys, Val, Phe, Ser, Ala, or Asp at position 163. In some embodiments, the modified CH3 domain polypeptide further comprises one, two, three, or four substitutions at positions including 153, 164, 165, and 188. In some embodiments, Trp, Tyr, Leu, or Gln may be present at position 153. In some embodiments, Ser, Thr, Gln, or Phe may be present at position 164. In some embodiments, Gln, Phe, or His may be present at position 165. In some embodiments, Gln may be present at position 188.

[0139] In certain embodiments, the modified CH3 domain polypeptide comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions selected from the following: Trp, Leu, or Glu at position 153; Tyr or Phe at position 157; Thr at position 159; Glu at position 160; Trp at position 161; Ser, Ala, Val, or Asn at position 162; Ser or Asn at position 163; Thr or Ser at position 186; Glu or Ser at position 188; Glu at position 189; and / or Phe at position 194. In some embodiments, the modified CH3 domain polypeptide contains all of the following eleven positions: Trp, Leu, or Glu at position 153; Tyr or Phe at position 157; Thr at position 159; Glu at position 160; Trp at position 161; Ser, Ala, Val, or Asn at position 162; Ser or Asn at position 163; Thr or Ser at position 186; Glu or Ser at position 188; Glu at position 189; and / or Phe at position 194.

[0140] In certain embodiments, the modified CH3 domain polypeptide comprises Leu or Met at position 157, Leu, His, or Pro at position 159, Val at position 160, Trp at position 161, Val or Ala at position 162, Pro at position 186, Thr at position 189, and / or Trp at position 194. In some embodiments, the modified CH3 domain polypeptide further comprises Ser, Thr, Gln, or Phe at position 164. In some embodiments, the modified CH3 domain polypeptide further comprises Trp, Tyr, Leu, or Gln at position 153 and / or Gln, Phe, or His at position 165. In some embodiments, Trp is present at position 153 and / or Gln is present at position 165. In some embodiments, the modified CH3 domain polypeptide does not have a Trp at position 153.

[0141] In other embodiments, the modified CH3 domain polypeptide comprises a Tyr at position 157, a Thr at position 159, a Glu or Val at position 160, a Trp at position 161, a Ser at position 162, a Ser or Thr at position 186, a Glu at position 189, and / or a Phe at position 194. In some embodiments, the modified CH3 domain polypeptide comprises a native Asn at position 163. In certain embodiments, the modified CH3 domain polypeptide further comprises a Trp, Tyr, Leu, or Gln at position 153 and / or a Glu at position 188. In some embodiments, the modified CH3 domain polypeptide further comprises a Trp at position 153 and / or a Glu at position 188.

[0142] In some embodiments, the modified CH3 domain comprises one or more of the following substitutions: Tyr at position 153, Thr at position 159, Trp at position 161, Val at position 162, Ser or Thr at position 186, Glu at position 188, and / or Phe at position 194.

[0143] In a further embodiment, the modified CH3 domain further comprises one, two, or three positions selected from the following: Lys, Arg, Gly, or Pro at position 187; Ser, Thr, Glu, or Lys at position 197; and Ser, Trp, or Gly at position 199.

[0144] In some embodiments, a modified CH3 domain polypeptide that specifically binds to a transferrin receptor has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 114-220 of any one of SEQ ID NOs: 4-29, 236-299, and 422-435. In some embodiments, such a modified CH3 domain polypeptide comprises amino acids 157-163 and / or 186-194 of any one of SEQ ID NOs: 4-29, 236-299, and 422-435. In some embodiments, such a modified CH3 domain polypeptide comprises amino acids 153-163 and / or 186-194 of any one of SEQ ID NOs: 4-29, 236-299, and 422-435. In some embodiments, the modified CH3 domain polypeptide comprises amino acids 153-163 and / or 186-199 of any one of SEQ ID NOs: 4-29, 236-299, and 422-435.

[0145] In some embodiments, a modified CH3 domain polypeptide that specifically binds to a transferrin receptor has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 114-220 of SEQ ID NO:1, with the proviso that the percent identity does not include the set of positions 157, 159, 160, 161, 162, 163, 186, 189, and 194. In some embodiments, a modified CH3 domain polypeptide comprises amino acids 157-163 and / or amino acids 186-194 set forth in any one of SEQ ID NOs:4-29, 236-299, and 422-435.

[0146] In some embodiments, the modified CH3 domain polypeptide has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOS: 4-29, 236-299, and 422-435, provided that at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the positions corresponding to positions 153, 157, 159, 160, 161, 162, 163, 164, 165, 186, 187, 188, 189, 194, 197, and 199 of any one of SEQ ID NOS: 4-29, 236-299, and 422-435 are not deleted or substituted.

[0147] In some embodiments, the modified CH3 domain polypeptide has at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs: 4-29, 236-299, and 422-435, and also contains one or more of the following amino acids: Trp, Tyr, Leu, Gln, or Glu at position 153; Leu, Tyr, Met, or Val at position 157; Leu, Thr, His, or Pro at position 159; Val, Pro, or an acidic amino acid at position 160; an aromatic amino acid, such as Trp, at position 161; Val, Ser, or Ala at position 162; at position 165; an acidic amino acid, Ala, Ser, Leu, Thr, or Pro at position 186; Lys, Arg, Gly, or Pro at position 187; Glu or Ser at position 188; Thr or an acidic amino acid at position 189; Trp, Tyr, His, or Phe at position 194; Ser, Thr, Glu, or Lys at position 197; and at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the following: Ser or Asn at position 3; Ser, Thr, Gln, or Phe at position 164; Gln, Phe, or His at position 165; an acidic amino acid, Ala, Ser, Leu, Thr, or Pro at position 186; Lys, Arg, Gly, or Pro at position 187;

[0148] In some embodiments, modified CH3 domain polypeptides of the invention have one or more substitutions at a set of amino acid positions including: 153, 157, 159, 160, 162, 163, 186, 188, 189, 194, 197, and 199, where the substitutions and positions are determined with reference to the sequence of SEQ ID NO: 13. In some embodiments, modified CH3 domains have one or more substitutions at position 153: Glu, Leu, Ser, Val, Trp, or Tyr; an aromatic amino acid (e.g., Tyr, Phe, or Trp), Met, Pro, or Val; an aromatic amino acid (e.g., Tyr, Phe, or Trp), Met, Pro, or Val; an aromatic amino acid (e.g., Tyr, Phe, or Trp), Met, Pro, or Val; an aromatic amino acid (e.g., Tyr, Phe, or Trp), Met, Pro, or Val; an aromatic amino acid (e.g., Ala, Ile, or Val), Ser, or Val; an aromatic amino acid (e.g., Ala, Ile, or Val), ... at position 163, Ser, Asn, Arg, or Thr at position 164, Thr, His, or Ser at position 186, Glu, Ser, Asp, Gly, Thr, Pro, Gln, or Arg at position 188, Glu or Arg at position 189, Phe, His, Lys, Tyr, or Trp at position 194, Ser, Thr, or Trp at position 197, and Ser, Cys, Pro, Met, or Trp at position 199. The modified CH3 domain polypeptide comprises the sequence: TIFF2025083406000001.tif19158 (wherein X1 is E, L, S, V, W, or Y; X2 is an aromatic amino acid (e.g., Y, F, or W), M, P, or V; X3 is T, N, or V; X4 is E, I, P, or V; X5 is an aliphatic amino acid (e.g., A, I, or V), S, or T; X6 is S, N, R, or T; X7 is T, H, or S; X8 is E, S, D, G, T, P, Q, or R; X9 is E or R; 10 is F, H, K, Y, or W, and X 11 is S, T, or W, and X 12X is S, C, P, M, or W. In certain embodiments, the modified CH3 domain polypeptide can have the sequence: XIWESX2GX3X4WX5X6 (SEQ ID NO:554), where XI is E, L, S, V, W, or Y, X is an aromatic amino acid (e.g., Y, F, or W), M, P, or V, X is T, N, or V, X is E, I, P, or V, X is an aliphatic amino acid (e.g., A, I, or V), S, or T, and X is S, N, R, or T. In certain embodiments, the modified CH3 domain polypeptide may comprise the sequence: X1KX2X3WQQGX4VFX5CX6 (SEQ ID NO:555), where X1 is T, H, or S; X2 is E, S, D, G, T, P, Q, or R; X3 is E or R; X4 is F, H, K, Y, or W; X5 is S, T, or W; and X6 is S, C, P, M, or W.

[0149] In some embodiments, the modified CH3 domain polypeptide comprises Glu, Leu, or Trp at position 153, an aromatic amino acid at position 157, Thr at position 159, Glu at position 160, an aliphatic amino acid or Ser at position 162, Ser or Asn at position 163, Thr or Ser at position 186, Glu or Ser at position 188, Glu at position 189, Phe, His, Tyr, or Trp at position 194, Ser at position 197, and Ser at position 199, where the substitutions and positions are determined with reference to SEQ ID NO:13. In a specific embodiment, the aromatic amino acid at position 157 is Tyr or Phe, and the aliphatic amino acid at position 162 is Ala or Val. The modified CH3 domain polypeptide comprises: TIFF2025083406000002.tif19158 (wherein X1 is E, L, or W, X2 is an aromatic amino acid (e.g., Y or F), X3 is T, X4 is E, X5 is an aliphatic amino acid (e.g., A or V) or S, X6 is S or N, X7 is T or S, X8 is E or S, X9 is E, and X 10is F, H, Y, or W, and X 11 is S and X 12 X is S). In certain embodiments, the modified CH3 domain polypeptide can have the sequence: X1WESX2GX3X4WX5X6 (SEQ ID NO:557) (wherein X1 is E, L, or W, X2 is an aromatic amino acid (e.g., Y or F), X3 is T, X4 is E, X5 is an aromatic amino acid (e.g., A or V) or S, and X6 is S or N). In certain embodiments, the modified CH3 domain polypeptide can have the sequence: X1KX2X3WQQGX4VFX5CX6 (SEQ ID NO:558) (wherein X1 is T or S, X2 is E or S, X3 is E, X4 is F, H, Y, or W, X5 is S, and X6 is S).

[0150] In further embodiments, the modified CH3 domain polypeptide can include Glu, Leu, or Trp at position 153, Tyr or Phe at position 157, Thr at position 159, Glu at position 160, Ala, Val, or Ser at position 162, Ser or Asn at position 163, Thr or Ser at position 186, Glu or Ser at position 188, Glu at position 189, Phe at position 194, Ser at position 197, and Ser at position 199, where the substitutions and positions are determined with reference to SEQ ID NO:13. TIFF2025083406000003.tif19158 (where X1 is E, L, or W, X2 is Y or F, X3 is T, X4 is E, X5 is S, A, or V, X6 is S or N, X7 is T or S, X8 is E or S, X9 is E, and X 10 is F and X 11 is S and X 12X1 is E, L, or W, X2 is Y or F, X3 is T, X4 is E, X5 is S, A, or V, and X6 is S or N. In certain embodiments, the modified CH3 domain polypeptide can comprise the sequence: X1KX2X3WQQGX4VFX5CX6 (SEQ ID NO:561) (wherein X1 is T or S, X2 is E or S, X3 is E, X4 is F, X5 is S, and X6 is S).

[0151] In some embodiments, a modified CH3 domain polypeptide of the invention comprises only one substitution at a set of amino acid positions including: 153, 157, 159, 160, 162, 163, 186, 188, 189, 194, 197, and 199, where the substitutions and positions are determined with reference to the sequence of SEQ ID NO:238. In some embodiments, the modified CH3 domain polypeptide comprises Glu, Leu, Ser, Val, Trp, or Tyr at position 153. The modified CH3 domain polypeptide can comprise Glu at position 153. The modified CH3 domain polypeptide can comprise Leu at position 153. The modified CH3 domain polypeptide can comprise Ser at position 153. The modified CH3 domain polypeptide can comprise Val at position 153. The modified CH3 domain polypeptide can comprise Trp at position 153. The modified CH3 domain polypeptide can comprise Tyr at position 153. In some embodiments, the modified CH3 domain polypeptide comprises Tyr, Phe, Trp, Met, Pro, or Val at position 157. The modified CH3 domain polypeptide can comprise Tyr at position 157. The modified CH3 domain polypeptide can comprise Phe at position 157. The modified CH3 domain polypeptide can comprise Trp at position 157. The modified CH3 domain polypeptide can comprise Met at position 157. The modified CH3 domain polypeptide can comprise Pro at position 157. The modified CH3 domain polypeptide can comprise Val at position 157. In some embodiments, the modified CH3 domain polypeptide comprises Thr, Asn, or Val at position 159. The modified CH3 domain polypeptide can comprise Thr at position 159. The modified CH3 domain polypeptide can comprise Asn at position 159. The modified CH3 domain polypeptide can comprise Val at position 159. In some embodiments, the modified CH3 domain polypeptide comprises Glu, Ile, Pro, or Val at position 160. The modified CH3 domain polypeptide can comprise a Glu at position 160. The modified CH3 domain polypeptide can comprise an Ile at position 160.The modified CH3 domain polypeptide can comprise Pro at position 160. The modified CH3 domain polypeptide can comprise Val at position 160. In some embodiments, the modified CH3 domain polypeptide comprises Ala, Ile, Val, Ser, or Thr at position 162. The modified CH3 domain polypeptide can comprise Ala at position 162. The modified CH3 domain polypeptide can comprise Ile at position 162. The modified CH3 domain polypeptide can comprise Val at position 162. The modified CH3 domain polypeptide can comprise Ser at position 162. The modified CH3 domain polypeptide can comprise Thr at position 162. In some embodiments, the modified CH3 domain polypeptide comprises Ser, Asn, Arg, or Thr at position 163. The modified CH3 domain polypeptide can comprise Ser at position 163. The modified CH3 domain polypeptide can comprise Asn at position 163. The modified CH3 domain polypeptide can comprise Arg at position 163. The modified CH3 domain polypeptide can comprise Thr at position 163. In some embodiments, the modified CH3 domain polypeptide comprises Thr, His, or Ser at position 186. The modified CH3 domain polypeptide can comprise Thr at position 186. The modified CH3 domain polypeptide can comprise His at position 186. The modified CH3 domain polypeptide can comprise Ser at position 186. In some embodiments, the modified CH3 domain polypeptide comprises Glu, Ser, Asp, Gly, Thr, Pro, Gln, or Arg at position 188. The modified CH3 domain polypeptide can comprise Glu at position 188. The modified CH3 domain polypeptide can comprise Ser at position 188. The modified CH3 domain polypeptide can comprise Asp at position 188. The modified CH3 domain polypeptide can comprise Gly at position 188. The modified CH3 domain polypeptide can comprise Thr at position 188. The modified CH3 domain polypeptide can comprise Pro at position 188. The modified CH3 domain polypeptide can comprise Gln at position 188.The modified CH3 domain polypeptide can comprise Arg at position 188. In some embodiments, the modified CH3 domain polypeptide comprises Glu or Arg at position 189. The modified CH3 domain polypeptide can comprise Glu at position 189. The modified CH3 domain polypeptide can comprise Arg at position 189. In some embodiments, the modified CH3 domain polypeptide comprises Phe, His, Lys, Tyr, or Trp at position 194. The modified CH3 domain polypeptide can comprise Phe at position 194. The modified CH3 domain polypeptide can comprise His at position 194. The modified CH3 domain polypeptide can comprise Lys at position 194. The modified CH3 domain polypeptide can comprise Tyr at position 194. The modified CH3 domain polypeptide can comprise Trp at position 194. In some embodiments, the modified CH3 domain polypeptide comprises Ser, Thr, or Trp at position 197. The modified CH3 domain polypeptide can comprise Ser at position 197. The modified CH3 domain polypeptide can comprise Thr at position 197. The modified CH3 domain polypeptide can comprise Trp at position 197. In some embodiments, the modified CH3 domain polypeptide comprises Ser, Cys, Pro, Met, or Trp at position 199. The modified CH3 domain polypeptide can comprise Ser at position 199. The modified CH3 domain polypeptide can comprise Cys at position 199. The modified CH3 domain polypeptide can comprise Pro at position 199. The modified CH3 domain polypeptide can comprise Met at position 199. The modified CH3 domain polypeptide can comprise Trp at position 199. The modified CH3 domain polypeptide can comprise the sequence of any one of SEQ ID NOs:563-574.

[0152] In some embodiments, modified CH3 domain polypeptides of the invention comprise one or more substitutions at a set of amino acid positions including: 153, 157, 159, 160, 162, 163, 164, 186, 189, and 194, where the substitutions and positions are determined relative to the sequence of SEQ ID NO:9. In some embodiments, the modified CH3 domain comprises Glu or Trp at position 153, Val, Trp, Leu, or Tyr at position 157, Leu, Pro, Phe, Thr, or His at position 159, Pro, Val, or Glu at position 160, Ala, Ser, Val, or Gly at position 162, Leu, His, Gln, Gly, Val, Ala, Asn, Asp, Thr, or Glu at position 163, Thr, Phe, Gln, Val, or Tyr at position 164, Leu, Ser, Glu, Ala, or Pro at position 186, Glu, Asp, Thr, or Asn at position 189, and Trp, Tyr, Phe, or His at position 194. The modified CH3 domain polypeptide has the sequence: TIFF2025083406000004.tif19159 (where X1 is E or W, X2 is V, W, L, or Y, X3 is L, P, F, T, or H, X4 is P, V, or E, X5 is A, S, V, or G, X6 is L, H, Q, G, V, AN, D, T, or E, X7 is T, F, Q, V, or Y, X8 is L, S, E, A, or P, X9 is E, D, T, or N, and X 10and X is W, Y, H, or F. In certain embodiments, the modified CH3 domain polypeptide can have the sequence: X1WESX2GX3X4WX5X6X7 (SEQ ID NO:575), where X1 is E or W, X2 is V, W, L, or Y, X3 is L, P, F, T, or H, X4 is P, V, or E, X5 is A, S, V, or G, X6 is L, H, Q, G, V, AN, D, T, or E, and X7 is T, F, Q, V, or Y. In certain embodiments, the modified CH3 domain polypeptide can have the sequence: X1KSX2WQQGX3 (SEQ ID NO:576), where X8 is L, S, E, A, or P, X9 is E, D, T, or N, and X 10 is W, Y, H, or F).

[0153] In some embodiments, the modified CH3 domain polypeptide comprises Glu or Trp at position 153, Trp, Leu, or Tyr at position 157, Thr or His at position 159, Val at position 160, Ala, Ser, or Val at position 162, Val, Asn, or Thr at position 163, Gln or Tyr at position 164, Pro at position 186, Thr or Asn at position 189, and Trp, Tyr, Phe, or His at position 194, where the substitutions and positions are determined with reference to SEQ ID NO:9. TIFF2025083406000005.tif19160 (wherein X1 is E or W, X2 is W, L, or Y, X3 is T or H, X4 is V, X5 is A, S, or V, X6 is V, T, or N, X7 is Y or Q, X8 is P, X9 is T or N, and X 10X is W, Y, H, or F. In certain embodiments, the modified CH3 domain polypeptide can have the sequence: X1WESX2GX3X4WX5X6X7 (SEQ ID NO:578), where X1 is E or W, X2 is W, L, or Y, X3 is T or H, X4 is V, X5 is A, S, or V, X6 is V, T, or N, and X7 is Y or Q. In certain embodiments, the modified CH3 domain polypeptide can have the sequence: X1KSX2WQQGX3 (SEQ ID NO:579), where X1 is P, X2 is T or N, and X3 is W, Y, H, or F.

[0154] In some embodiments, the transferrin receptor binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:116-130. In other embodiments, the transferrin receptor binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:116-130, but with one or two amino acid substitutions in the sequence. In some embodiments, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:116-130, but with three amino acid substitutions in the sequence.

[0155] In some embodiments, the transferrin receptor binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:131-139. In other embodiments, the transferrin receptor binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:131-130, but with one or two amino acid substitutions in the sequence. In some embodiments, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:131-139, but with three or four amino acid substitutions in the sequence.

[0156] In some embodiments, the transferrin receptor binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:303-339. In other embodiments, the transferrin receptor binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:303-339, but with one or two amino acid substitutions in the sequence. In some embodiments, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:303-339, but with three amino acid substitutions in the sequence.

[0157] In some embodiments, the transferrin receptor binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 136, 138, and 340-345. In other embodiments, the transferrin receptor binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 136, 138, and 340-345, but with one or two amino acid substitutions in the sequence. In some embodiments, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 136, 138, and 340-345, but with three or four amino acid substitutions in the sequence.

[0158] In further embodiments, the transferrin receptor binding polypeptide comprises amino acids 157-194, amino acids 153-194, or amino acids 153-199 of any one of SEQ ID NOs: 4-29, 236-299, and 422-435. In further embodiments, the polypeptide comprises an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical to amino acids 157-194 of any one of SEQ ID NOs: 4-29, 236-299, and 422-435, or to amino acids 153-194 or amino acids 153-199 of any one of SEQ ID NOs: 4-29, 236-299, and 422-435.

[0159] In some embodiments, the polypeptide comprises any one of SEQ ID NOS: 4-29, 236-299, and 422-435. In further embodiments, the polypeptide comprises any one of SEQ ID NOS: 4-29, 236-299, and 422-435 without the first three amino acid residues "PCP" at the amino terminus. In further embodiments, the polypeptide can have at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOS: 4-29, 236-299, and 422-435 determined without the first three amino acid residues "PCP" at the amino terminus.

[0160] Group (ii) that binds to the transferrin receptor of CH3: 118, 119, 120, 122, 210, 211, 212, and 213 In some embodiments, modified CH3 domain polypeptides according to the invention comprise at least three or at least four, typically five, six, seven, or eight substitutions at a set of amino acid positions including 118, 119, 120, 122, 210, 211, 212, and 213 (set ii). Exemplary substitutions that can be introduced at these positions are shown in Table 5. In some embodiments, modified CH3 domain polypeptides comprise a Gly at position 210, a Phe at position 211, and / or an Asp at position 213. In some embodiments, a Glu can be present at position 213. In certain embodiments, modified CH3 domain polypeptides comprise at least one substitution at the following positions: That is, Phe or lie at position 118, Asp, Glu, Gly, Ala, or Lys at position 119, Tyr, Met, Leu, lie, or Asp at position 120, Thr or Ala at position 122, Gly at position 210, Phe at position 211, His, Tyr, Ser, or Phe at position 212, or Asp at position 213. In some embodiments, two, three, four, five, six, seven, or all eight of positions 118, 119, 120, 122, 210, 211, 212, and 213 have a substitution as specified in this paragraph. In some embodiments, the modified CH3 domain polypeptide can include conservative substitutions of particular amino acids at one or more positions within the above sets, e.g., amino acids within groups based on the same charge, hydrophobicity, side chain ring structure (e.g., aromatic amino acids), or size, and / or polarity or nonpolarity.

[0161] In some embodiments, a modified CH3 domain polypeptide that specifically binds to a transferrin receptor has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 114-220 of any one of SEQ ID NOs:30-46. In some embodiments, such a modified CH3 domain polypeptide comprises amino acids 118-122 and / or amino acids 210-213 of any one of SEQ ID NOs:30-46.

[0162] In some embodiments, the modified CH3 domain polypeptide has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 114-220 of SEQ ID NO:1, with the proviso that the percent identity does not include the set of positions 118, 119, 120, 122, 210, 211, 212, and 213. In some embodiments, the modified CH3 domain polypeptide comprises amino acids 118-122 and / or amino acids 210-213 set forth in any one of SEQ ID NOs:30-46.

[0163] In some embodiments, the transferrin receptor binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 140-153. In other embodiments, the transferrin receptor binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 140-153, but with one or two amino acid substitutions in the sequence.

[0164] In some embodiments, the transferrin receptor binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 154 to 157. In other embodiments, the transferrin receptor binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 154 to 157, but with one amino acid substitution or two amino acid substitutions within the sequence.

[0165] In further embodiments, the transferrin receptor binding polypeptide comprises amino acids 118-213 of any one of SEQ ID NOs: 30-46. In further embodiments, the polypeptide can comprise an amino acid sequence having at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 118-213 of any one of SEQ ID NOs: 30-46.

[0166] In some embodiments, the polypeptide comprises any one of SEQ ID NOs:30-46. In further embodiments, the polypeptide comprises any one of SEQ ID NOs:30-46 without the first three amino acid residues "PCP" at the amino terminus. In further embodiments, the polypeptide can have at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs:30-46 or any one of SEQ ID NOs:30-46 determined without the first three amino acid residues "PCP" at the amino terminus.

[0167] CH2 Transferrin Receptor Binding Polypeptide In some embodiments, the domain to be modified is a human Ig CH2 domain, such as an IgG CH2 domain. The CH2 domain may be derived from any IgG subtype, i.e., IgG1, IgG2, IgG3, or IgG4. In the context of IgG antibodies, the CH2 domain refers to the segment of amino acids from about 231 to about 340, when numbered according to the EU numbering scheme. For purposes of identifying a set of corresponding amino acid positions for transferrin receptor binding, positions within the CH2 domain are determined relative to SEQ ID NO:2 or relative to amino acids 4-113 of SEQ ID NO:1. Substitutions are also determined relative to SEQ ID NO:1, i.e., an amino acid is considered a substitution for the amino acid at the corresponding position in SEQ ID NO:1. SEQ ID NO:1 includes a partial hinge region sequence, PCP, as amino acids 1-3. Although these three residues are not part of the Fc region, the numbering of the positions within the CH2 domain with reference to SEQ ID NO:1 includes these first three amino acids.

[0168] As noted above, sets of residues in a CH2 domain that can be modified in accordance with the present invention are numbered herein with reference to SEQ ID NO:1. For example, any CH2 domain, such as the CH2 domain of IgG1, IgG2, IgG3, or IgG4, can have modifications (e.g., amino acid substitutions) at one or more sets of residues that correspond to the residues at the recited positions in SEQ ID NO:1. An alignment of the amino acid sequence of human IgG1 of SEQ ID NO:1 with human IgG2, IgG3, and IgG4 is shown in Figure 38. The respective positions in the IgG2, IgG3, and IgG4 sequences that correspond to any particular position in SEQ ID NO:1 can be readily determined.

[0169] In one embodiment, a modified CH2 domain polypeptide that specifically binds to the transferrin receptor binds to an epitope within the apical domain of the transferrin receptor. The sequence of the apical domain of the human transferrin receptor is set forth in SEQ ID NO:107, which corresponds to amino acids 198-378 of the uniprotein sequence P02786 of human transferrin receptor 1. The modified CH2 domain polypeptide can bind to the transferrin receptor without blocking or otherwise inhibiting transferrin binding to the receptor. In some embodiments, binding of transferrin to TfR is not substantially inhibited. In some embodiments, binding of transferrin to TfR is inhibited by less than about 50% (e.g., less than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%). In some embodiments, binding of transferrin to TfR is inhibited by less than about 20% (e.g., less than about 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%).

[0170] Group (iii) that binds to the transferrin receptor of CH2: 47, 49, 56, 58, 59, 60, 61, 62, and 63 In some embodiments, modified CH2 domain polypeptides of the invention contain at least three or at least four, and typically five, six, seven, eight, or nine substitutions at a set of amino acid positions including positions 47, 49, 56, 58, 59, 60, 61, 62, and 63 (set iii). Exemplary substitutions that can be introduced at these positions are shown in Table 1. In some embodiments, modified CH2 domain polypeptides contain Glu at position 60 and / or Trp at position 61. In some embodiments, the modified CH2 domain polypeptide comprises at least one substitution at the following positions: Glu, Gly, Gln, Ser, Ala, Asn, Tyr, or Trp at position 47; Ile, Val, Asp, Glu, Thr, Ala, or Tyr at position 49; Asp, Pro, Met, Leu, Ala, Asn, or Phe at position 56; Arg, Ser, Ala, or Gly at position 58; Tyr, Trp, Arg, or Val at position 59; Glu at position 60; Trp or Tyr at position 61; Gln, Tyr, His, Ile, Phe, Val, or Asp at position 62; or Leu, Trp, Arg, Asn, Tyr, or Val at position 63. In some embodiments, two, three, four, five, six, seven, eight, or all nine of positions 47, 49, 56, 58, 59, 60, 61, 62, and 63 have substitutions as specified in this paragraph. In some embodiments, the modified CH2 domain polypeptide can include conservative substitutions of particular amino acids at one or more of the positions in the above sets, e.g., amino acids in groups based on the same charge, hydrophobicity, side chain ring structure (e.g., aromatic amino acids), or size, and / or polar or nonpolar.

[0171] In some embodiments, the modified CH2 domain polypeptide comprises Glu, Gly, Gln, Ser, Ala, Asn, or Tyr at position 47, He, Val, Asp, Glu, Thr, Ala, or Tyr at position 49, Asp, Pro, Met, Leu, Ala, or Asn at position 56, Arg, Ser, or Ala at position 58, Tyr, Trp, Arg, or Val at position 59, Glu at position 60, Trp at position 61, Gln, Tyr, His, He, Phe, or Val at position 62, and / or Leu, Trp, Arg, Asn, or Tyr at position 63. In some embodiments, the modified CH2 domain polypeptide comprises Arg at position 58, Tyr or Trp at position 59, Glu at position 60, Trp at position 61, and / or Asp or Trp at position 63.

[0172] In some embodiments, a modified CH2 domain polypeptide that specifically binds to a transferrin receptor has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 4-113 of any one of SEQ ID NOs: 47-62. In some embodiments, such a modified CH2 domain polypeptide comprises amino acids 47-49 and / or amino acids 56-63 of any one of SEQ ID NOs: 47-62.

[0173] In some embodiments, modified CH2 domain polypeptides of the invention have at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 4-113 of SEQ ID NO:1, with the proviso that the percent identity does not include the set of positions 47, 49, 56, 58, 59, 60, 61, 62, and 63. In some embodiments, modified CH2 domain polypeptides comprise amino acids 47-49 and / or amino acids 56-63 set forth in any one of SEQ ID NOs:47-62.

[0174] In some embodiments, the transferrin receptor binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 158-171. In other embodiments, the transferrin receptor binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 158-171, except that one amino acid in the sequence has been substituted.

[0175] In some embodiments, the transferrin receptor binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:172-186. In other embodiments, the transferrin receptor binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:172-186, but with one amino acid substitution or with two amino acid substitutions in the sequence. In other embodiments, the transferrin receptor binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:172-186, but with three or four amino acid substitutions in the sequence.

[0176] In further embodiments, the transferrin receptor binding polypeptide comprises amino acids 47-63 of any one of SEQ ID NOs: 47-62. In further embodiments, the polypeptide can comprise an amino acid sequence having at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 47-63 of any one of SEQ ID NOs: 47-63.

[0177] In some embodiments, the polypeptide comprises any one of SEQ ID NOs:47-62. In further embodiments, the polypeptide comprises any one of SEQ ID NOs:47-62 without the first three amino acid residues "PCP" at the amino terminus. In further embodiments, the polypeptide can have at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs:47-62 determined without the first three amino acid residues "PCP" at the amino terminus, or to any one of SEQ ID NOs:47-62.

[0178] Group (iv) that binds to the transferrin receptor of CH2: 39, 40, 41, 42, 43, 44, 68, 70, 71, and 72 In some embodiments, modified CH2 domain polypeptides of the invention contain at least three or at least four, and typically five, six, seven, eight, nine, or ten, substitutions at a set of amino acid positions including positions 39, 40, 41, 42, 43, 44, 68, 70, 71, and 72 (set iv). Exemplary substitutions that can be introduced at these positions are shown in Table 2. In some embodiments, modified CH2 domain polypeptides contain Pro at position 43, Glu at position 68, and / or Tyr at position 70. In some embodiments, the modified CH2 domain polypeptide comprises at least one substitution at the following positions: Pro, Phe, Ala, Met, or Asp at position 39; Gln, Pro, Arg, Lys, Ala, Ile, Leu, Glu, Asp, or Tyr at position 40; Thr, Ser, Gly, Met, Val, Phe, Trp, or Leu at position 41; Pro, Val, Ala, Thr, or Asp at position 42; Pro, Val, or Phe at position 43; Trp, Gln, Thr, or Glu at position 44; Glu, Val, Thr, Leu, or Trp at position 68; Tyr, His, Val, or Asp at position 70; Thr, His, Gln, Arg, Asn, or Val at position 71; or Tyr, Asn, Asp, Ser, or Pro at position 72. In some embodiments, two, three, four, five, six, seven, eight, nine, or all ten of positions 39, 40, 41, 42, 43, 44, 68, 70, 71, and 72 have a substitution as specified in this paragraph. In some embodiments, the modified CH2 domain polypeptide can include conservative substitutions of particular amino acids at one or more of the positions in the above sets, e.g., amino acids in groups based on the same charge, hydrophobicity, side chain ring structure (e.g., aromatic amino acids), or size, and / or polar or nonpolar.

[0179] In some embodiments, the modified CH2 domain polypeptide comprises Pro, Phe, or Ala at position 39, Gln, Pro, Arg, Lys, Ala, or Ile at position 40, Thr, Ser, Gly, Met, Val, Phe, or Trp at position 41, Pro, Val, or Ala at position 42, Pro at position 43, Trp or Gln at position 44, Glu at position 68, Tyr at position 70, Thr, His, or Gln at position 71, and / or Tyr, Asn, Asp, or Ser at position 72.

[0180] In some embodiments, the modified CH2 domain polypeptide comprises Met at position 39, Leu or Glu at position 40, Trp at position 41, Pro at position 42, Val at position 43, Thr at position 44, Val or Thr at position 68, His at position 70, His, Arg, or Asn at position 71, and / or Pro at position 72.

[0181] In some embodiments, the modified CH2 domain polypeptide comprises an Asp at position 39, an Asp at position 40, a Leu at position 41, a Thr at position 42, a Phe at position 43, a Gln at position 44, a Val or Leu at position 68, a Val at position 70, a Thr at position 71, and / or a Pro at position 72.

[0182] In some embodiments, a modified CH2 domain polypeptide that specifically binds to a transferrin receptor has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 4-113 of any one of SEQ ID NOs:63-85. In some embodiments, such a modified CH2 domain polypeptide comprises amino acids 39-44 and / or amino acids 68-72 of any one of SEQ ID NOs:63-85.

[0183] In some embodiments, modified CH2 domain polypeptides of the invention have at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 4-113 of SEQ ID NO:1, with the proviso that the percent identity does not include the set of positions 39, 40, 41, 42, 43, 44, 68, 70, 71, and 72. In some embodiments, modified CH2 domain polypeptides comprise amino acids 39-44 and / or amino acids 68-72 set forth in any one of SEQ ID NOs:63-85.

[0184] In some embodiments, the transferrin receptor binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:187-204. In other embodiments, the transferrin receptor binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:187-204, but with one or two amino acid substitutions in the sequence. In other embodiments, the transferrin receptor binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:187-204, but with three amino acid substitutions in the sequence.

[0185] In some embodiments, the transferrin receptor binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:205-215. In other embodiments, the transferrin receptor binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:205-215, but with one amino acid substitution or two amino acid substitutions within the sequence.

[0186] In further embodiments, the transferrin receptor binding polypeptide comprises amino acids 39-72 of any one of SEQ ID NOs:63-85. In further embodiments, the polypeptide comprises an amino acid sequence having at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 39-72 of any one of SEQ ID NOs:63-85.

[0187] In some embodiments, the polypeptide comprises any one of SEQ ID NOs:63-85. In further embodiments, the polypeptide comprises any one of SEQ ID NOs:63-85 without the first three amino acid residues "PCP" at the amino terminus. In further embodiments, the polypeptide can have at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs:63-85 or any one of SEQ ID NOs:63-85 determined without the first three amino acid residues "PCP" at the amino terminus.

[0188] CH2 transferrin receptor binding group (v): 41, 42, 43, 44, 45, 65, 66, 67, 69, and 73 In some embodiments, modified CH2 domain polypeptides of the invention contain at least three or at least four, and typically five, six, seven, eight, nine, or ten, substitutions at a set of amino acid positions that includes positions 41, 42, 43, 44, 45, 65, 66, 67, 69, and 73 (set v). Exemplary substitutions that can be introduced at these positions are shown in Table 3. In some embodiments, the modified CH2 domain polypeptide comprises at least one substitution at the following positions: Val or Asp at position 41, Pro, Met, or Asp at position 42, Pro or Trp at position 43, Arg, Trp, Glu, or Thr at position 44, Met, Tyr, or Trp at position 45, Leu or Trp at position 65, Thr, Val, Ile, or Lys at position 66, Ser, Lys, Ala, or Leu at position 67, His, Leu, or Pro at position 69, or Val or Trp at position 73. In some embodiments, two, three, four, five, six, seven, eight, nine, or all ten of positions 41, 42, 43, 44, 45, 65, 66, 67, 69, and 73 have a substitution as specified in this paragraph. In some embodiments, the modified CH2 domain polypeptide can include conservative substitutions of particular amino acids at one or more positions within the above sets, e.g., amino acids within groups based on the same charge, hydrophobicity, side chain ring structure (e.g., aromatic amino acids), or size, and / or polarity or nonpolarity.

[0189] In some embodiments, the modified CH2 domain polypeptide comprises Val at position 41, Pro at position 42, Pro at position 43, Arg or Trp at position 44, Met at position 45, Leu at position 65, Thr at position 66, Ser at position 67, His at position 69, and / or Val at position 73.

[0190] In some embodiments, the modified CH2 domain polypeptide comprises Asp at position 41, Met or Asp at position 42, Trp at position 43, Glu or Thr at position 44, Tyr or Trp at position 45, Trp at position 65, Val, Ile, or Lys at position 66, Lys, Ala, or Leu at position 67, Leu or Pro at position 69, and / or Trp at position 73.

[0191] In some embodiments, a modified CH2 domain polypeptide that specifically binds to a transferrin receptor has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 4-113 of any one of SEQ ID NOs: 86-90. In some embodiments, such a modified CH3 domain polypeptide comprises amino acids 41-45 and / or amino acids 65-73 of any one of SEQ ID NOs: 86-90.

[0192] In some embodiments, modified CH2 domain polypeptides of the invention have at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 4-113 of SEQ ID NO:1, with the proviso that the percent identity does not include the set of positions 41, 42, 43, 44, 45, 65, 66, 67, 69, and 73. In some embodiments, modified CH2 domain polypeptides comprise amino acids 41-45 and / or amino acids 65-73 set forth in any one of SEQ ID NOs:86-90.

[0193] In some embodiments, the transferrin receptor binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 216-220. In other embodiments, the transferrin receptor binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 216-220, but with one or two amino acid substitutions in the sequence.

[0194] In some embodiments, the transferrin receptor binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:221-224. In other embodiments, the transferrin receptor binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:221-224, but with one amino acid substitution or with two amino acid substitutions in the sequence. In other embodiments, the transferrin receptor binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:221-224, but with three or four amino acid substitutions in the sequence.

[0195] In further embodiments, the transferrin receptor binding polypeptide comprises amino acids 41-73 of any one of SEQ ID NOs: 86-90. In further embodiments, the polypeptide can comprise a sequence having at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 41-73 of any one of SEQ ID NOs: 86-90.

[0196] In some embodiments, the polypeptide comprises any one of SEQ ID NOs:86-90. In further embodiments, the polypeptide comprises any one of SEQ ID NOs:86-90 without the first three amino acid residues "PCP" at the amino terminus. In further embodiments, the polypeptide can have at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs:86-90 determined without the first three amino acid residues "PCP" at the amino terminus, or to any one of SEQ ID NOs:86-90.

[0197] Group (vi) that binds to the transferrin receptor of CH2: 45, 47, 49, 95, 97, 99, 102, 103, and 104 In some embodiments, modified CH2 domain polypeptides of the invention comprise at least three or at least four, and typically five, six, seven, eight, or nine substitutions at a set of amino acid positions including: 45, 47, 49, 95, 97, 99, 102, 103, and 104 (set vi). Exemplary substitutions that can be introduced at these positions are shown in Table 4. In some embodiments, the modified CH2 domain polypeptide comprises a Trp at position 103. In some embodiments, the modified CH2 domain polypeptide comprises at least one substitution at the following positions: Trp, Val, Ile, or Ala at position 45; Trp or Gly at position 47; Tyr, Arg, or Glu at position 49; Ser, Arg, or Gln at position 95; Val, Ser, or Phe at position 97; Ile, Ser, or Trp at position 99; Trp, Thr, Ser, Arg, or Asp at position 102; Trp at position 103; and Ser, Lys, Arg, or Val at position 104. In some embodiments, two, three, four, five, six, seven, eight, or all nine of positions 45, 47, 49, 95, 97, 99, 102, 103, and 104 have a substitution as specified in this paragraph. In some embodiments, the modified CH2 domain polypeptide can include conservative substitutions of particular amino acids at one or more positions within the above sets, e.g., amino acids within groups based on the same charge, hydrophobicity, side chain ring structure (e.g., aromatic amino acids), or size, and / or polarity or nonpolarity.

[0198] In some embodiments, the modified CH2 domain polypeptide comprises two, three, four, five, six, seven, eight, or nine positions selected from the following: Trp, Val, Ile, or Ala at position 45; Trp or Gly at position 47; Tyr, Arg, or Glu at position 49; Ser, Arg, or Gln at position 95; Val, Ser, or Phe at position 97; Ile, Ser, or Trp at position 99; Trp, Thr, Ser, Arg, or Asp at position 102; Trp at position 103; and Ser, Lys, Arg, or Val at position 104.

[0199] In some embodiments, the modified CH2 domain polypeptide comprises Val or Ile at position 45, Gly at position 47, Arg at position 49, Arg at position 95, Ser at position 97, Ser at position 99, Thr, Ser, or Arg at position 102, Trp at position 103, and / or Lys or Arg at position 104.

[0200] In some embodiments, a modified CH2 domain polypeptide that specifically binds to a transferrin receptor has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 4-113 of any one of SEQ ID NOs:91-95. In some embodiments, such a modified CH3 domain polypeptide comprises amino acids 45-49 and / or amino acids 95-104 of any one of SEQ ID NOs:91-95.

[0201] In some embodiments, modified CH2 domain polypeptides of the invention have at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 4-113 of SEQ ID NO:1, with the proviso that the percent identity does not include the set of positions 45, 47, 49, 95, 97, 99, 102, 103, and 104. In some embodiments, modified CH2 domain polypeptides comprise amino acids 45-49 and / or amino acids 95-104 set forth in any one of SEQ ID NOs:91-95.

[0202] In some embodiments, the transferrin receptor binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 225-228. In other embodiments, the transferrin receptor binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 225-228, but with one or two amino acid substitutions in the sequence.

[0203] In some embodiments, the transferrin receptor binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:229-233. In other embodiments, the transferrin receptor binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:229-223, but with one amino acid substitution or with two amino acid substitutions in the sequence. In other embodiments, the transferrin receptor binding polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:229-233, but with three, four, or five amino acid substitutions in the sequence.

[0204] In further embodiments, the transferrin receptor binding polypeptide comprises amino acids 45-104 of any one of SEQ ID NOs:91-95. In further embodiments, the polypeptide can comprise a sequence having at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 45-104 of any one of SEQ ID NOs:91-95.

[0205] In some embodiments, the polypeptide comprises any one of SEQ ID NOs:91-95. In further embodiments, the polypeptide comprises any one of SEQ ID NOs:91-95 without the first three amino acid residues "PCP" at the amino terminus. In further embodiments, the polypeptide can have at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs:91-95 determined without the first three amino acid residues "PCP" at the amino terminus, or to any one of SEQ ID NOs:91-95.

[0206] Exemplary Polypeptides Comprising Modified CH3 or CH2 Domains The modified CH3 or CH2 domain polypeptides of the present invention can be linked to another domain of the Fc region. In some embodiments, the modified CH3 domain polypeptides of the present invention can be linked to a CH2 domain, which can be a naturally occurring CH2 domain or a mutant CH2 domain that is normally C-terminal to the CH2 domain. In some embodiments, the modified CH2 domain polypeptides of the present invention can be linked to a CH3 domain, which can be a naturally occurring CH3 domain or a mutant CH3 domain that is normally N-terminal to the CH3 domain. In some embodiments, a polypeptide comprising a modified CH2 domain linked to a CH3 domain or a polypeptide comprising a modified CH3 domain linked to a CH2 domain further comprises a partial or complete hinge region of an antibody, thereby forming the modified CH3 domain polypeptide or modified CH2 domain polypeptide as part of an Fc region with a partial or complete hinge region. The hinge region can be from any immunoglobulin subclass or isotype. An exemplary immunoglobulin hinge is an IgG hinge region, such as the IgG1 hinge region (e.g., the amino acid sequence of the human IgG1 hinge, EPKSCDKTHTCPPCP (SEQ ID NO:234)). In further embodiments, the polypeptide, which may be in an Fc format with a hinge or partial hinge region, is further linked to another moiety, such as a Fab fragment, to create an Fc-Fab fusion that binds to the transferrin receptor. In some embodiments, the Fc-Fab fusion that binds to the transferrin receptor further comprises a modified CH3 domain polypeptide or a modified CH2 domain polypeptide, a hinge region, and a Fab fragment. The Fab fragment can be directed to any target of interest, such as a therapeutic neurological target, where the Fab is delivered to the target by transcytosis across the blood-brain barrier mediated by binding of the modified CH3 domain polypeptide or modified CH2 domain polypeptide to the transferrin receptor.

[0207] In some embodiments, the Fab fragment linked to the transferrin receptor binding polypeptide is capable of binding to a tau protein (e.g., human tau protein) or a fragment thereof, hi some embodiments, the Fab fragment is capable of binding to phosphorylated tau protein, unphosphorylated tau protein, splice isoforms of tau protein, N-terminally truncated tau protein, C-terminally truncated tau protein, and / or fragments thereof.

[0208] In some embodiments, the Fab fragment linked to the transferrin receptor binding polypeptide is capable of binding to a beta-secretase 1 (BACE1) protein (e.g., a human BACE1 protein) or a fragment thereof. In some embodiments, the Fab fragment is capable of binding to one or more splice isoforms of the BACE1 protein or a fragment thereof.

[0209] In some embodiments, the Fab fragment linked to the transferrin receptor binding polypeptide is capable of binding to a triggering receptor 2 (TREM2) protein expressed on myeloid cells (e.g., a human TREM2 protein) or a fragment thereof.

[0210] In some embodiments, the Fab fragment linked to the transferrin receptor binding polypeptide is capable of binding to an α-synuclein protein (e.g., a human α-synuclein protein) or a fragment thereof, hi some embodiments, the Fab fragment is capable of binding to monomeric α-synuclein, oligomeric α-synuclein, α-synuclein fibrils, soluble α-synuclein, and / or fragments thereof.

[0211] In some embodiments, an Fc-Fab fusion comprising a modified CH2 or CH3 domain polypeptide of the invention is a subunit of a dimer. In some embodiments, the dimer is a heterodimer. In some embodiments, the dimer is a homodimer. In some embodiments, the dimer comprises one polypeptide that binds to the transferrin receptor (i.e., is monovalent for binding to the transferrin receptor). In some embodiments, the dimer comprises a second polypeptide that binds to the transferrin receptor. The second polypeptide may comprise the same modified CH3 domain polypeptide (or modified CH2 domain polypeptide) present in the Fc-Fab fusion, thereby providing a bivalent binding homodimer, or a second modified CH3 domain polypeptide (or modified CH2 domain polypeptide) of the invention may provide a second transferrin receptor binding site. In some embodiments, the dimer comprises a first subunit comprising a modified CH3 domain polypeptide or modified CH2 domain polypeptide, and a second subunit comprising CH2 and CH3 domains, neither of which binds to the transferrin receptor.

[0212] The transferrin receptor binding polypeptide may be fused to a different polypeptide of interest other than the Fab. For example, in some embodiments, the transferrin receptor binding polypeptide may be fused to a different polypeptide that is desirable for targeting to transferrin receptor-expressing cells or for delivery across the endothelium, e.g., the blood-brain barrier, by transcytosis. In some embodiments, the transferrin receptor binding polypeptide is fused to a soluble protein, e.g., the extracellular domain of the receptor or a growth factor, cytokine, or enzyme.

[0213] In still other embodiments, transferrin receptor binding polypeptides can be fused to peptides or proteins useful for protein purification, such as polyhistidine, epitope tags such as FLAG, c-Myc, hemagglutinin tags, glutathione S-transferase (GST), thioredoxin, protein A, protein G, or maltose binding protein (MBP). Optionally, the peptide or protein fused to the transferrin receptor binding polypeptide can contain a protease cleavage site, such as a cleavage site for factor Xa or thrombin.

[0214] Transferrin receptor-binding polypeptides of the invention can have a wide range of binding affinities based, for example, on the format of the polypeptide. For example, in some embodiments, polypeptides comprising a modified CH3 domain or a modified CH2 domain have transferrin receptor binding affinities ranging from 1 pM to 10 μM. In some embodiments, affinity can be measured in a monovalent format. In other embodiments, affinity can be measured in a bivalent format, for example, as a dimer comprising a polypeptide-Fab fusion protein.

[0215] Methods for analyzing binding affinity, binding kinetics, and cross-reactivity are well known in the art. These methods include, but are not limited to, solid-phase binding assays (e.g., ELISA assays), immunoprecipitation, surface plasmon resonance (e.g., Biacore™ (GE Healthcare, Piscataway, NJ)), kinetic exclusion assays (e.g., KinExA®), flow cytometry, fluorescence-activated cell sorting (FACS), biolayer interferometry (e.g., Octet® (ForteBio, Inc., Menlo Park, CA)), and Western blot analysis. In some embodiments, ELISA is used to determine binding affinity and / or cross-reactivity. Methods for performing ELISA assays are well known in the art and are also described in the Examples section below. In some embodiments, surface plasmon resonance (SPR) is used to determine binding affinity, binding kinetics, and / or cross-reactivity. In some embodiments, kinetic exclusion assays are used to determine binding affinity, binding kinetics, and / or cross-reactivity. In some embodiments, biolayer interferometry assays are used to determine binding affinity, binding kinetics, and / or cross-reactivity.

[0216] Further mutations within the Fc region containing modified CH3 or CH2 domain polypeptides The polypeptides provided herein that are modified to bind the transferrin receptor and initiate transport across the BBB may contain additional mutations, e.g., to increase serum stability, modulate effector function, affect glycosylation, reduce immunogenicity in humans, and / or enable knob-and-hole heterodimerization of the polypeptide.

[0217] In some embodiments, polypeptides according to the invention have at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to a corresponding wild-type Fc region (e.g., an Fc region of human IgG1, IgG2, IgG3, or IgG4).

[0218] Polypeptides of the invention may have other mutations introduced outside of the designated set of amino acids, e.g., to affect glycosylation, increase serum half-life, or, in the case of CH3 domains, to enable knob-and-hole heterodimerization of polypeptides comprising the modified CH3 domain. Generally, this method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding hole ("hole") at the interface of a second polypeptide, such that the protrusion can locate within the hole, promoting heterodimer formation and preventing homodimer formation. The protrusion is formed by replacing a small amino acid side chain at the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A complementary hole of the same or similar size as the protrusion is formed at the interface of the second polypeptide by replacing the large amino acid side chain with a smaller one (e.g., alanine or threonine). Such additional mutations are introduced at positions within the polypeptide that do not negatively affect binding of the modified CH3 or CH2 domain to the transferrin receptor.

[0219] In one exemplary embodiment of a knobs-and-holes approach to dimerization, a first Fc polypeptide subunit to be dimerized has a tryptophan instead of the native threonine at a position corresponding to position 139 of SEQ ID NO:1, and a second Fc polypeptide subunit of the dimer has a valine instead of the native tyrosine at a position corresponding to position 180 of SEQ ID NO:1. The second subunit of the Fc polypeptide may further comprise a substitution of serine for the native threonine at a position corresponding to position 139 of SEQ ID NO:1, and an alanine for the native leucine at a position corresponding to position 141 of SEQ ID NO:1.

[0220] The polypeptides described herein can also be engineered to have other modifications for heterodimerization (e.g., electrostatic manipulation of contact residues within the naturally charged CH3-CH3 interface, or hydrophobic patch modifications, etc.).

[0221] In some embodiments, modifications can be introduced to increase serum half-life. For example, in some embodiments, the Fc region comprises a CH2 domain comprising a Tyr at position corresponding to position 25 of SEQ ID NO:1, a Thr at position corresponding to position 27 of SEQ ID NO:1, and a Glu at position corresponding to position 29 of SEQ ID NO:1.

[0222] In some embodiments, mutations, e.g., substitutions, are introduced at one or more of positions 17-30, 52-57, 80-90, 156-163, and 201-208 relative to SEQ ID NO:1. In some embodiments, one or more mutations are introduced at positions 24, 25, 27, 28, 29, 80, 81, 82, 84, 85, 87, 158, 159, 160, 162, 201, 206, 207, or 209 relative to SEQ ID NO:1. In some embodiments, mutations are introduced at one, two, or three of positions 25, 27, and 29 relative to SEQ ID NO:1. In some embodiments, the mutations are M25Y, S27T, and T29E when numbered relative to SEQ ID NO:1. In some embodiments, the polypeptides described herein further comprise the mutations M25Y, S27T, and T29E. In some embodiments, the mutations are introduced at one or two of positions 201 and 207 when numbered relative to SEQ ID NO:1. In some embodiments, the mutations are M201L and N207S when numbered relative to SEQ ID NO:1. In some embodiments, the polypeptides described herein further comprise the mutation N207S with or without M201L. In some embodiments, the polypeptides described herein comprise substitutions at one, two, or all three of positions T80, E153, and N207 when numbered relative to SEQ ID NO:1. In some embodiments, the mutations are T80Q and N207A. In some embodiments, the polypeptides described herein comprise mutations T80A, E153A, and N207A. In some embodiments, the polypeptides described herein comprise substitutions at positions T23 and M201, when numbered relative to SEQ ID NO:1. In some embodiments, the polypeptides described herein comprise mutations T23Q and M201L. In some embodiments, the polypeptides described herein comprise substitutions at positions M201 and N207, when numbered relative to SEQ ID NO:1.In some embodiments, the polypeptides described herein comprise the substitutions M201L and N207S. In some embodiments, the polypeptides described herein comprise the substitutions N207S or N207A.

[0223] Fc effector functions In some embodiments, Fc regions comprising modified CH2 or CH3 domains have effector function (i.e., these Fc regions have the ability to induce a specific biological function upon binding to an Fc receptor expressed on an effector cell that mediates that function.) Effector cells include, but are not limited to, monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and cytotoxic T cells.

[0224] Examples of antibody effector functions include, but are not limited to, C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. Effector functions can vary depending on the antibody class. For example, native human IgG1 and IgG3 antibodies can induce ADCC and CDC activity upon binding to the appropriate Fc receptor present on immune system cells, and native human IgG1, IgG2, IgG3, and IgG4 antibodies can induce ADCP function upon binding to the appropriate Fc receptor present on immune cells.

[0225] In some embodiments, the polypeptides described herein can include additional modifications that reduce effector function, or in some embodiments, polypeptides comprising modified CH2 or CH3 domains of the invention can include additional modifications that enhance effector function.

[0226] Exemplary Fc polypeptide mutations that modulate effector function include, but are not limited to, substitutions within the CH2 domain, for example, substitutions at positions corresponding to positions 7 and 8 of SEQ ID NO:1. In some embodiments, the substitutions within the modified CH2 domain comprise Ala at positions 7 and 8 of SEQ ID NO:1. In some embodiments, the substitutions within the modified CH2 domain comprise Ala at positions 7 and 8 and Gly at position 102 of SEQ ID NO:1.

[0227] Additional Fc polypeptide mutations that modulate effector function include, but are not limited to, one or more substitutions at positions 238, 265, 269, 270, 297, 327, and 329 (which, in the EU numbering scheme, correspond to positions 11, 38, 42, 43, 70, 100, and 102 when numbered based on SEQ ID NO:1). Exemplary substitutions (when numbered using the EU numbering scheme) include the following: Position 329 can be mutated to replace proline with glycine or arginine, or an amino acid residue large enough to disrupt the Fc / Fcγ receptor interface formed between proline 329 of the Fc and tryptophan residues Trp87 and Trp110 of the FcγRIII. Further exemplary substitutions include S228P, E233P, L235E, N297A, N297D, and P331S. Multiple substitutions may be present, such as L234A and L235A in the Fc region of human IgG1, L234A, L235A, and P329G in the Fc region of human IgG1, S228P and L235E in the Fc region of human IgG4, L234A and G237A in the Fc region of human IgG1, L234A, L235A, and G237A in the Fc region of human IgG1, V234A and G237A in the Fc region of human IgG2, L235A, G237A, and E318A in the Fc region of human IgG4, and S228P and L236E in the Fc region of human IgG4. In some embodiments, polypeptides of the invention may have one or more amino acid substitutions that modulate ADCC (e.g., substitutions at positions 298, 333, and / or 334 in the Fc region according to the EU numbering scheme).

[0228] In some embodiments, the polypeptides described herein may have one or more amino acid substitutions that increase or decrease ADCC, or may have mutations that alter C1q binding and / or CDC.

[0229] Exemplary Polypeptides Containing Additional Mutations The polypeptides described herein (e.g., any one of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.23.2.1, and CH3C.35.23.1.1) may contain knob mutations (e.g., T139W when numbered relative to SEQ ID NO:1), hole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), effector function-modulating mutations (e.g., T139S, T141A, and T180V when numbered relative to SEQ ID NO:1), and / or effector function-modulating mutations (e.g., T139S, T141A, and T180V when numbered relative to SEQ ID NO:1). The antibody may include additional mutations, including L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1, and / or mutations that enhance serum stability (e.g., (i) M25Y, S27T, and T29E when numbered relative to SEQ ID NO:1, or (ii) N207S with or without M201L when numbered relative to SEQ ID NO:1).

[0230] In some embodiments, a polypeptide described herein (e.g., any one of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.23.2.1, and CH3C.35.23.1.1) can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs:4-95, 236-299, and 422-435. In some embodiments, a polypeptide having the sequence of any one of SEQ ID NOs: 4-95, 236-299, and 422-435 can be modified to have a knob mutation.

[0231] In some embodiments, a polypeptide described herein (e.g., any one of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.23.2.1, and CH3C.35.23.1.1) comprises a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), and a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1). The polypeptide may have at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs: 4-95, 236-299, and 422-435. In some embodiments, a polypeptide having a sequence of any one of SEQ ID NOs: 4-95, 236-299, and 422-435 can be engineered to have knob mutations and mutations that modulate effector function.

[0232] In some embodiments, a polypeptide described herein (e.g., any one of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.23.2.1, and CH3C.35.23.1.1) comprises a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), a serum stability enhancing mutation (e.g., (i) M25Y, S27T, and T29E when numbered relative to SEQ ID NO:1, or (ii) N207S with or without M201L when numbered relative to SEQ ID NO:1), and a mutation associated with SEQ ID NO:1. The polypeptide may have at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs: 4-95, 236-299, and 422-435. In some embodiments, a polypeptide having a sequence of any one of SEQ ID NOs: 4-95, 236-299, and 422-435 can be engineered to have knob mutations and mutations that enhance serum stability.

[0233] In some embodiments, the polypeptides described herein (e.g., any one of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.23.2.1, and CH3C.35.23.1.1) comprise a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), a mutation that enhances serum stability (e.g., (i) the amino acid sequence of SEQ ID NO:1), a mutation that enhances ATP synthesis (e.g., (ii) the amino acid sequence of SEQ ID NO:1), a mutation that enhances ATP synthesis (e.g., (iii) the amino acid sequence of SEQ ID NO:1), a mutation that enhances ATP synthesis (e.g., (iv) the amino acid sequence of SEQ ID NO:1), a mutation that enhances ATP synthesis (e.g., (v) the amino acid sequence of SEQ ID NO:1), a mutation that enhances ATP synthesis (e.g., (v) the amino acid sequence of SEQ ID NO:1), a mutation that enhances ATP synthesis (e.g., (vi ...v) the amino acid sequence of SEQ ID NO:1), a mutation that enhances ATP synthesis (e.g., (vi) the amino acid sequence of SEQ or (ii) N207S with or without M201L when numbered relative to SEQ ID NO:1), and any one of SEQ ID NOs:4-95, 236-299, and 422-435. In some embodiments, a polypeptide having a sequence of any one of SEQ ID NOs:4-95, 236-299, and 422-435 can be engineered to have knob mutations, mutations that modulate effector function, and mutations that increase serum stability.

[0234] In some embodiments, a polypeptide described herein (e.g., any one of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.23.2.1, and CH3C.35.23.1.1) can have a hole mutation (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs:4-95, 236-299, and 422-435. In some embodiments, a polypeptide having the sequence of any one of SEQ ID NOs: 4-95, 236-299, and 422-435 can be modified to have a hole mutation.

[0235] In some embodiments, a polypeptide described herein (e.g., any one of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.23.2.1, and CH3C.35.23.1.1) comprises a hole mutation (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), and a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1). The polypeptide may have at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs: 4-95, 236-299, and 422-435. In some embodiments, a polypeptide having any one of SEQ ID NOs: 4-95, 236-299, and 422-435 can be engineered to have a hole mutation and a mutation that modulates effector function.

[0236] In some embodiments, the polypeptides described herein (e.g., any one of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.23.2.1, and CH3C.35.23.1.1) contain a hole mutation (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), a mutation that enhances serum stability (e.g., (i) M25Y, S27T, and T29E when numbered relative to SEQ ID NO:1, or (ii) ... The polypeptide may have at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOS: 4-95, 236-299, and 422-435 (N207S with or without M201L, when numbered relative to SEQ ID NOS: 1), and any one of SEQ ID NOS: 4-95, 236-299, and 422-435. In some embodiments, a polypeptide having the sequence of any one of SEQ ID NOS: 4-95, 236-299, and 422-435 can be modified to contain a hole mutation and a mutation that enhances serum stability.

[0237] In some embodiments, the polypeptides described herein (e.g., any one of clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, CH3C.35.23, CH3C.35.21, CH3C.35.20.1.1, CH3C.23.2.1, and CH3C.35.23.1.1) comprise any of a wide variety of mutations, including, but not limited to, whole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), mutations that enhance serum stability (e.g., (i) any of the mutations in SEQ ID NO:1, (ii) any of the mutations in SEQ ID NO:1, (iii) any of the mutations in SEQ ID NO:1, (iv) any of the mutations in SEQ ID NO:1, (v) any of the mutations in SEQ ID NO:1, (vi ... or (ii) N207S with or without M201L when numbered relative to SEQ ID NO:1), and any one of SEQ ID NOs:4-95, 236-299, and 422-435. In some embodiments, a polypeptide having a sequence of any one of SEQ ID NOs:4-95, 236-299, and 422-435 can be engineered to contain hole mutations, mutations that modulate effector function, and mutations that increase serum stability.

[0238] Clone CH3C.35.20.1 In some embodiments, clone CH3C.35.20.1 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:349. In some embodiments, clone CH3C.35.20.1 with the knob mutation has the sequence of SEQ ID NO:349.

[0239] In some embodiments, clone CH3C.35.20.1 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:350 or 351. In some embodiments, clone CH3C.35.20.1 having a knob mutation and a mutation that modulates effector function has the sequence of SEQ ID NO:350 or 351.

[0240] In some embodiments, clone CH3C.35.20.1 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), serum stability-enhancing mutations (e.g., M25Y, S27T, and T29E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:352. In some embodiments, clone CH3C.35.20.1 with knob mutations and serum stability-enhancing mutations has the sequence of SEQ ID NO:352.

[0241] In some embodiments, clone CH3C.35.20.1 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), a serum stability-enhancing mutation (e.g., N207S with or without M201L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:485. In some embodiments, clone CH3C.35.20.1 with a knob mutation and a serum stability-enhancing mutation has the sequence of SEQ ID NO:485.

[0242] In some embodiments, clone CH3C.35.20.1 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), a mutation that increases serum stability (e.g., M25Y, S27T, and T29E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:353 or 354. In some embodiments, clone CH3C.35.20.1 having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability has the sequence of SEQ ID NO:353 or 354.

[0243] In some embodiments, clone CH3C.35.20.1 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), a mutation that enhances serum stability (e.g., N207S with or without M201L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:486 or 487. In some embodiments, clone CH3C.35.20.1 having a knob mutation, a mutation that modulates effector function, and a mutation that enhances serum stability has the sequence of SEQ ID NO:486 or 487.

[0244] In some embodiments, clone CH3C.35.20.1 can have hole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:355. In some embodiments, clone CH3C.35.20.1 with hole mutations has the sequence of SEQ ID NO:355.

[0245] In some embodiments, clone CH3C.35.20.1 can have hole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:356 or 357. In some embodiments, clone CH3C.35.20.1 having hole mutations and mutations that modulate effector function has the sequence of SEQ ID NO:356 or 357.

[0246] In some embodiments, clone CH3C.35.20.1 can have whole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), mutations that increase serum stability (e.g., M25Y, S27T, and T29E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:358. In some embodiments, clone CH3C.35.20.1 with whole mutations and mutations that increase serum stability has the sequence of SEQ ID NO:358.

[0247] In some embodiments, clone CH3C.35.20.1 can have whole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), mutations that increase serum stability (e.g., N207S with or without M201L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:488. In some embodiments, clone CH3C.35.20.1 with whole mutations and mutations that increase serum stability has the sequence of SEQ ID NO:488.

[0248] In some embodiments, clone CH3C.35.20.1 can have whole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability (e.g., M25Y, S27T, and T29E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:359 or 360. In some embodiments, clone CH3C.35.20.1 having whole mutations, mutations that modulate effector function, and mutations that increase serum stability has the sequence of SEQ ID NO:359 or 360.

[0249] In some embodiments, clone CH3C.35.20.1 can have whole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability (e.g., N207S with or without M201L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:489 or 490. In some embodiments, clone CH3C.35.20.1 having whole mutations, mutations that modulate effector function, and mutations that increase serum stability has the sequence of SEQ ID NO:489 or 490.

[0250] Clone CH3C.35.23.2 In some embodiments, clone CH3C.35.23.2 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:361. In some embodiments, clone CH3C.35.23.2 with the knob mutation has the sequence of SEQ ID NO:361.

[0251] In some embodiments, clone CH3C.35.23.2 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:362 or 363. In some embodiments, clone CH3C.35.23.2 with a knob mutation and a mutation that modulates effector function has the sequence of SEQ ID NO:362 or 363.

[0252] In some embodiments, clone CH3C.35.23.2 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), serum stability-enhancing mutations (e.g., M25Y, S27T, and T29E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:364. In some embodiments, clone CH3C.35.23.2 with knob mutations and serum stability-enhancing mutations has the sequence of SEQ ID NO:364.

[0253] In some embodiments, clone CH3C.35.23.2 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), a serum stability-enhancing mutation (e.g., N207S with or without M201L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:492. In some embodiments, clone CH3C.35.23.2 with a knob mutation and a serum stability-enhancing mutation has the sequence of SEQ ID NO:492.

[0254] In some embodiments, clone CH3C.35.23.2 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), a mutation that increases serum stability (e.g., M25Y, S27T, and T29E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:365 or 366. In some embodiments, clone CH3C.35.23.2 having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability has the sequence of SEQ ID NO:365 or 366.

[0255] In some embodiments, clone CH3C.35.23.2 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), a mutation that enhances serum stability (e.g., N207S with or without M201L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:493 or 494. In some embodiments, clone CH3C.35.23.2 having a knob mutation, a mutation that modulates effector function, and a mutation that enhances serum stability has the sequence of SEQ ID NO:493 or 494.

[0256] In some embodiments, clone CH3C.35.23.2 can have hole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:367. In some embodiments, clone CH3C.35.23.2 with hole mutations has the sequence of SEQ ID NO:367.

[0257] In some embodiments, clone CH3C.35.23.2 can have hole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:368 or 369. In some embodiments, clone CH3C.35.23.2 with hole mutations and mutations that modulate effector function has the sequence of SEQ ID NO:368 or 369.

[0258] In some embodiments, clone CH3C.35.23.2 can have whole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), mutations that increase serum stability (e.g., M25Y, S27T, and T29E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:370. In some embodiments, clone CH3C.35.23.2 with whole mutations and mutations that increase serum stability has the sequence of SEQ ID NO:370.

[0259] In some embodiments, clone CH3C.35.23.2 can have whole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), mutations that increase serum stability (e.g., N207S with or without M201L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:495. In some embodiments, clone CH3C.35.23.2 with whole mutations and mutations that increase serum stability has the sequence of SEQ ID NO:495.

[0260] In some embodiments, clone CH3C.35.23.2 can have whole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability (e.g., M25Y, S27T, and T29E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:371 or 372. In some embodiments, clone CH3C.35.23.2 having whole mutations, mutations that modulate effector function, and mutations that increase serum stability has the sequence of SEQ ID NO:371 or 372.

[0261] In some embodiments, clone CH3C.35.23.2 can have whole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability (e.g., N207S with or without M201L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:496 or 497. In some embodiments, clone CH3C.35.23.2 having whole mutations, mutations that modulate effector function, and mutations that increase serum stability has the sequence of SEQ ID NO:496 or 497.

[0262] Clone CH3C.35.23.3 In some embodiments, clone CH3C.35.23.3 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:373. In some embodiments, clone CH3C.35.23.3 with the knob mutation has the sequence of SEQ ID NO:373.

[0263] In some embodiments, clone CH3C.35.23.3 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:374 or 375. In some embodiments, clone CH3C.35.23.3 with a knob mutation and a mutation that modulates effector function has the sequence of SEQ ID NO:374 or 375.

[0264] In some embodiments, clone CH3C.35.23.3 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), serum stability-enhancing mutations (e.g., M25Y, S27T, and T29E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:376. In some embodiments, clone CH3C.35.23.3 with knob mutations and serum stability-enhancing mutations has the sequence of SEQ ID NO:376.

[0265] In some embodiments, clone CH3C.35.23.3 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), a serum stability-enhancing mutation (e.g., N207S with or without M201L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:499. In some embodiments, clone CH3C.35.23.3 with a knob mutation and a serum stability-enhancing mutation has the sequence of SEQ ID NO:499.

[0266] In some embodiments, clone CH3C.35.23.3 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), a mutation that increases serum stability (e.g., M25Y, S27T, and T29E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:377 or 378. In some embodiments, clone CH3C.35.23.3 having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability has the sequence of SEQ ID NO:377 or 378.

[0267] In some embodiments, clone CH3C.35.23.3 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), a mutation that enhances serum stability (e.g., N207S with or without M201L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:500 or 501. In some embodiments, clone CH3C.35.23.3 having a knob mutation, a mutation that modulates effector function, and a mutation that enhances serum stability has the sequence of SEQ ID NO:500 or 501.

[0268] In some embodiments, clone CH3C.35.23.3 can have hole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:379. In some embodiments, clone CH3C.35.23.3 with hole mutations has the sequence of SEQ ID NO:379.

[0269] In some embodiments, clone CH3C.35.23.3 can have hole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:380 or 381. In some embodiments, clone CH3C.35.23.3 with hole mutations and mutations that modulate effector function has the sequence of SEQ ID NO:380 or 381.

[0270] In some embodiments, clone CH3C.35.23.3 can have whole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), mutations that increase serum stability (e.g., M25Y, S27T, and T29E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:382. In some embodiments, clone CH3C.35.23.3 with whole mutations and mutations that increase serum stability has the sequence of SEQ ID NO:382.

[0271] In some embodiments, clone CH3C.35.23.3 can have whole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), mutations that increase serum stability (e.g., N207S with or without M201L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:502. In some embodiments, clone CH3C.35.23.3 with whole mutations and mutations that increase serum stability has the sequence of SEQ ID NO:502.

[0272] In some embodiments, clone CH3C.35.23.3 can have whole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability (e.g., M25Y, S27T, and T29E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:383 or 384. In some embodiments, clone CH3C.35.23.3 having whole mutations, mutations that modulate effector function, and mutations that increase serum stability has the sequence of SEQ ID NO:383 or 384.

[0273] In some embodiments, clone CH3C.35.23.3 can have whole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability (e.g., N207S with or without M201L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:503 or 504. In some embodiments, clone CH3C.35.23.3 having whole mutations, mutations that modulate effector function, and mutations that increase serum stability has the sequence of SEQ ID NO:503 or 504.

[0274] Clone CH3C.35.23.4 In some embodiments, clone CH3C.35.23.4 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:385. In some embodiments, clone CH3C.35.23.4 with the knob mutation has the sequence of SEQ ID NO:385.

[0275] In some embodiments, clone CH3C.35.23.4 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:386 or 387. In some embodiments, clone CH3C.35.23.4 with a knob mutation and a mutation that modulates effector function has the sequence of SEQ ID NO:386 or 387.

[0276] In some embodiments, clone CH3C.35.23.4 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), serum stability-enhancing mutations (e.g., M25Y, S27T, and T29E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:388. In some embodiments, clone CH3C.35.23.4 with knob mutations and serum stability-enhancing mutations has the sequence of SEQ ID NO:388.

[0277] In some embodiments, clone CH3C.35.23.4 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), a serum stability-enhancing mutation (e.g., N207S with or without M201L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:506. In some embodiments, clone CH3C.35.23.4 with a knob mutation and a serum stability-enhancing mutation has the sequence of SEQ ID NO:506.

[0278] In some embodiments, clone CH3C.35.23.4 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), a mutation that increases serum stability (e.g., M25Y, S27T, and T29E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:389 or 390. In some embodiments, clone CH3C.35.23.4 with a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability has the sequence of SEQ ID NO:389 or 390.

[0279] In some embodiments, clone CH3C.35.23.4 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), a mutation that enhances serum stability (e.g., N207S with or without M201L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:507 or 508. In some embodiments, clone CH3C.35.23.4 having a knob mutation, a mutation that modulates effector function, and a mutation that enhances serum stability has the sequence of SEQ ID NO:507 or 508.

[0280] In some embodiments, clone CH3C.35.23.4 can have hole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:391. In some embodiments, clone CH3C.35.23.4 with hole mutations has the sequence of SEQ ID NO:391.

[0281] In some embodiments, clone CH3C.35.23.4 can have hole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:392 or 393. In some embodiments, clone CH3C.35.23.4 with hole mutations and mutations that modulate effector function has the sequence of SEQ ID NO:392 or 393.

[0282] In some embodiments, clone CH3C.35.23.4 can have whole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), mutations that increase serum stability (e.g., M25Y, S27T, and T29E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:394. In some embodiments, clone CH3C.35.23.4 with whole mutations and mutations that increase serum stability has the sequence of SEQ ID NO:394.

[0283] In some embodiments, clone CH3C.35.23.4 can have whole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), mutations that increase serum stability (e.g., N207S with or without M201L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:509. In some embodiments, clone CH3C.35.23.4 with whole mutations and mutations that increase serum stability has the sequence of SEQ ID NO:509.

[0284] In some embodiments, clone CH3C.35.23.4 can have whole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability (e.g., M25Y, S27T, and T29E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:395 or 396. In some embodiments, clone CH3C.35.23.4 having whole mutations, mutations that modulate effector function, and mutations that increase serum stability has the sequence of SEQ ID NO:395 or 396.

[0285] In some embodiments, clone CH3C.35.23.4 can have whole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability (e.g., N207S with or without M201L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:510 or 511. In some embodiments, clone CH3C.35.23.4 having whole mutations, mutations that modulate effector function, and mutations that increase serum stability has the sequence of SEQ ID NO:510 or 511.

[0286] Clone CH3C.35.21.17.2 In some embodiments, clone CH3C.35.21.17.2 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:397. In some embodiments, clone CH3C.35.21.17.2 with the knob mutation has the sequence of SEQ ID NO:397.

[0287] In some embodiments, clone CH3C.35.21.17.2 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:398 or 399. In some embodiments, clone CH3C.35.21.17.2 having a knob mutation and a mutation that modulates effector function has the sequence of SEQ ID NO:398 or 399.

[0288] In some embodiments, clone CH3C.35.21.17.2 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), serum stability-enhancing mutations (e.g., M25Y, S27T, and T29E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:400. In some embodiments, clone CH3C.35.21.17.2 with knob mutations and serum stability-enhancing mutations has the sequence of SEQ ID NO:400.

[0289] In some embodiments, clone CH3C.35.21.17.2 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), a serum stability-enhancing mutation (e.g., N207S with or without M201L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:513. In some embodiments, clone CH3C.35.21.17.2 with a knob mutation and a serum stability-enhancing mutation has the sequence of SEQ ID NO:513.

[0290] In some embodiments, clone CH3C.35.21.17.2 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), a mutation that increases serum stability (e.g., M25Y, S27T, and T29E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:401 or 402. In some embodiments, clone CH3C.35.21.17.2 having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability has the sequence of SEQ ID NO:401 or 402.

[0291] In some embodiments, clone CH3C.35.21.17.2 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), a mutation that enhances serum stability (e.g., N207S with or without M201L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:514 or 515. In some embodiments, clone CH3C.35.21.17.2 having a knob mutation, a mutation that modulates effector function, and a mutation that enhances serum stability has the sequence of SEQ ID NO:514 or 515.

[0292] In some embodiments, clone CH3C.35.21.17.2 can have hole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:403. In some embodiments, clone CH3C.35.21.17.2 with hole mutations has the sequence of SEQ ID NO:403.

[0293] In some embodiments, clone CH3C.35.21.17.2 can have hole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:404 or 405. In some embodiments, clone CH3C.35.21.17.2 with hole mutations and mutations that modulate effector function has the sequence of SEQ ID NO:404 or 405.

[0294] In some embodiments, clone CH3C.35.21.17.2 can have whole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), mutations that increase serum stability (e.g., M25Y, S27T, and T29E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:406. In some embodiments, clone CH3C.35.21.17.2 with whole mutations and mutations that increase serum stability has the sequence of SEQ ID NO:406.

[0295] In some embodiments, clone CH3C.35.21.17.2 can have whole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), mutations that increase serum stability (e.g., N207S with or without M201L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:516. In some embodiments, clone CH3C.35.21.17.2 with whole mutations and mutations that increase serum stability has the sequence of SEQ ID NO:516.

[0296] In some embodiments, clone CH3C.35.21.17.2 can have whole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability (e.g., M25Y, S27T, and T29E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:407 or 408. In some embodiments, clone CH3C.35.21.17.2 having whole mutations, mutations that modulate effector function, and mutations that increase serum stability has the sequence of SEQ ID NO:407 or 408.

[0297] In some embodiments, clone CH3C.35.21.17.2 can have whole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability (e.g., N207S with or without M201L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:517 or 518. In some embodiments, clone CH3C.35.21.17.2 having whole mutations, mutations that modulate effector function, and mutations that increase serum stability has the sequence of SEQ ID NO:517 or 518.

[0298] Clone CH3C.35.23 In some embodiments, clone CH3C.35.23 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:409. In some embodiments, clone CH3C.35.23 with the knob mutation has the sequence of SEQ ID NO:409.

[0299] In some embodiments, clone CH3C.35.23 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:410 or 411. In some embodiments, clone CH3C.35.23 with a knob mutation and a mutation that modulates effector function has the sequence of SEQ ID NO:410 or 411.

[0300] In some embodiments, clone CH3C.35.23 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), serum stability-enhancing mutations (e.g., M25Y, S27T, and T29E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:412. In some embodiments, clone CH3C.35.23 with the knob mutation and the serum stability-enhancing mutation has the sequence of SEQ ID NO:412.

[0301] In some embodiments, clone CH3C.35.23 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), a serum stability-enhancing mutation (e.g., N207S with or without M201L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:520. In some embodiments, clone CH3C.35.23 with a knob mutation and a serum stability-enhancing mutation has the sequence of SEQ ID NO:520.

[0302] In some embodiments, clone CH3C.35.23 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), a mutation that increases serum stability (e.g., M25Y, S27T, and T29E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:413 or 414. In some embodiments, clone CH3C.35.23 having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability has the sequence of SEQ ID NO:413 or 414.

[0303] In some embodiments, clone CH3C.35.23 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), a mutation that increases serum stability (e.g., N207S with or without M201L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:521 or 522. In some embodiments, clone CH3C.35.23 having a knob mutation, a mutation that modulates effector function, and a mutation that increases serum stability has the sequence of SEQ ID NO:521 or 522.

[0304] In some embodiments, clone CH3C.35.23 can have hole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:415. In some embodiments, clone CH3C.35.23 with hole mutations has the sequence of SEQ ID NO:415.

[0305] In some embodiments, clone CH3C.35.23 can have hole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:416 or 417. In some embodiments, clone CH3C.35.23 with hole mutations and mutations that modulate effector function has the sequence of SEQ ID NO:416 or 417.

[0306] In some embodiments, clone CH3C.35.23 can have whole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), mutations that increase serum stability (e.g., M25Y, S27T, and T29E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:418. In some embodiments, clone CH3C.35.23 with whole mutations and mutations that increase serum stability has the sequence of SEQ ID NO:418.

[0307] In some embodiments, clone CH3C.35.23 can have whole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), mutations that increase serum stability (e.g., N207S with or without M201L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:523. In some embodiments, clone CH3C.35.23 with whole mutations and mutations that increase serum stability has the sequence of SEQ ID NO:523.

[0308] In some embodiments, clone CH3C.35.23 can have whole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability (e.g., M25Y, S27T, and T29E when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:419 or 420. In some embodiments, clone CH3C.35.23 having whole mutations, mutations that modulate effector function, and mutations that increase serum stability has the sequence of SEQ ID NO:419 or 420.

[0309] In some embodiments, clone CH3C.35.23 can have whole mutations (e.g., T139S, L141A, and Y180V when numbered relative to SEQ ID NO:1), mutations that modulate effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), mutations that increase serum stability (e.g., N207S with or without M201L when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:524 or 525. In some embodiments, clone CH3C.35.23 having whole mutations, mutations that modulate effector function, and mutations that increase serum stability has the sequence of SEQ ID NO:524 or 525.

[0310] Clone CH3C.35.21 In some embodiments, clone CH3C.35.21 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1) and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:436. In some embodiments, clone CH3C.35.21 with the knob mutation has the sequence of SEQ ID NO:436.

[0311] In some embodiments, clone CH3C.35.21 can have a knob mutation (e.g., T139W when numbered relative to SEQ ID NO:1), a mutation that modulates effector function (e.g., L7A, L8A, and / or P102G (e.g., L7A and L8A) when numbered relative to SEQ ID NO:1), and at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of SEQ ID NO:437 or 438. In some embodiments, clone CH3C.35.21 with a knob mutation and a mutation that modulates effector...

Claims

[Claim 1] The invention described herein.

Images